Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production

ABSTRACT

Isolated nucleic acid molecules, designated SMP nucleic acid molecules, which encode novel SMP proteins from  Corynebacterium glutamicum  are described. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing SMP nucleic acid molecules, and host cells into which the expression vectors have been introduced. The invention still further provides isolated SMP proteins, mutated SMP proteins, fusion proteins, antigenic peptides and methods for the improvement of production of a desired compound from  C. glutamicum  based on genetic engineering of SMP genes in this organism.

RELATED APPLICATIONS

This application is a continuation of prior U.S. patent application Ser.No. 09/602,740, filed Jun. 23, 2000, which claims priority to U.S.Provisional Patent Application Ser. No. 60/141,031, filed Jun. 25, 1999,U.S. Provisional Patent Application Ser. No. 60/143,208, filed Jul. 9,1999, and U.S. Provisional Patent Application Ser. No. 60/151,572, filedAug. 31, 1999. This application also claims priority to prior GermanPatent Application No. 19931412.8, filed Jul. 8, 1999, German PatentApplication No. 19931413.6, filed Jul. 8, 1999, German PatentApplication No. 19931419.5, filed Jul. 8, 1999, German PatentApplication No. 19931420.9, filed Jul. 8, 1999, German PatentApplication No. 19931424.1, filed Jul. 8, 1999, German PatentApplication No. 19931428.4, filed Jul. 8, 1999, German PatentApplication No. 19931431.4, filed Jul. 8, 1999, German PatentApplication No. 19931433.0, filed Jul. 8, 1999, German PatentApplication No. 19931434.9, filed Jul. 8, 1999, German PatentApplication No. 19931510.8, filed Jul. 8, 1999, German PatentApplication No. 19931562.0, filed Jul. 8, 1999, German PatentApplication No. 19931634.1, filed Jul. 8, 1999, German PatentApplication No. 19932180.9, filed Jul. 9, 1999, German PatentApplication No. 19932227.9, filed Jul. 9, 1999, German PatentApplication No. 19932230.9, filed Jul. 9, 1999, German PatentApplication No. 19932924.9, filed Jul. 14, 1999, German PatentApplication No. 19932973.7, filed Jul. 14, 1999, German PatentApplication No. 19933005.0, filed Jul. 14, 1999, German PatentApplication No. 19940765.7, filed Aug. 27, 1999, German PatentApplication No. 19942076.9, filed Sep. 3, 1999, German PatentApplication No. 19942079.3, filed Sep. 3, 1999, German PatentApplication No. 19942086.6, filed Sep. 3, 1999, German PatentApplication No. 19942087.4, filed Sep. 3, 1999, German PatentApplication No. 19942088.2, filed Sep. 3, 1999, German PatentApplication No. 19942095.5, filed Sep. 3, 1999, German PatentApplication No. 19942123.4, filed Sep. 3, 1999, and German PatentApplication No. 19942125.0, filed Sep. 3, 1999. The entire contents ofall of the aforementioned applications are hereby expressly incorporatedherein by this reference.

INCORPORATION OF MATERIAL SUBMITTED ON COMPACT DISCS

This application incorporates herein by reference the material containedon the compact discs snbmitted herewith as part of this application.Specifically, the file “seqlist.txt” (2859 kB) contained on each of Copy1, Copy 2 and the CRF copy of the Scquence Listing is herebyincorporated herein by reference. This file was created on Feb. 17,2004.

BACKGROUND OF THE INVENTION

Certain products and by-products of naturally-occurring metabolicprocesses in cells have utility in a wide array of industries, includingthe food, feed, cosmetics, and pharmaceutical industries. Thesemolecules, collectively termed ‘fine chemicals’, include organic acids,both proteinogenic and non-proteinogenic amino acids, nucleotides andnucleosides, lipids and fatty acids, diols, carbohydrates, aromaticcompounds, vitamins and cofactors, and enzymes. Their production is mostconveniently performed through the large-scale culture of bacteriadeveloped to produce and secrete large quantities of one or more desiredmolecules. One particularly useful organism for this purpose isCorynebacterium glutamicum, a gram positive, nonpathogenic bacterium.Through strain selection, a number of mutant strains have been developedwhich produce an array of desirable compounds. However, selection ofstrains improved for the production of a particular molecule is atime-consuming and difficult process.

SUMMARY OF THE INVENTION

The invention provides novel bacterial nucleic acid molecules which havea variety of uses. These uses include the identification ofmicroorganisms which can be used to produce fine chemicals, themodulation of fine chemical production in C. glutamicum or relatedbacteria, the typing or identification of C. glutamicum or relatedbacteria, as reference points for mapping the C. glutamicum genome, andas markers for transformation. These novel nucleic acid molecules encodeproteins, referred to herein as sugar metabolism and oxidativephosphorylation (SMP) proteins.

C. glutamicum is a gram positive, aerobic bacterium which is commonlyused in industry for the large-scale production of a variety of finechemicals, and also for the degradation of hydrocarbons (such as inpetroleum spills) and for the oxidation of terpenoids. The SMP nucleicacid molecules of the invention, therefore, can be used to identifymicroorganisms which can be used to produce fine chemicals, e.g., byfermentation processes. Modulation of the expression of the SMP nucleicacids of the invention, or modification of the sequence of the SMPnucleic acid molecules of the invention, can be used to modulate theproduction of one or more fine chemicals from a microorganism (e.g., toimprove the yield or production of one or more fine chemicals from aCorynebacterium or Brevibacterium species).

The SMP nucleic acids of the invention may also be used to identify anorganism as being Corynebacterium glutamicum or a close relativethereof, or to identify the presence of C. glutamicum or a relativethereof in a mixed population of microorganisms. The invention providesthe nucleic acid sequences of a number of C. glutamicum genes; byprobing the extracted genomic DNA of a culture of a unique or mixedpopulation of microorganisms under stringent conditions with a probespanning a region of a C. glutamicum gene which is unique to thisorganism, one can ascertain whether this organism is present. AlthoughCorynebacterium glutamicum itself is nonpathogenic, it is related tospecies pathogenic in humans, such as Corynebacterium diphtheriae (thecausative agent of diphtheria); the detection of such organisms is ofsignificant clinical relevance.

The SMP nucleic acid molecules of the invention may also serve asreference points for mapping of the C. glutamicum genome, or of genomesof related organisms. Similarly, these molecules, or variants orportions thereof, may serve as markers for genetically engineeredCorynebacterium or Brevibacterium species. e.g.e.g. The SMP proteinsencoded by the novel nucleic acid molecules of the invention are capableof, for example, performing a function involved in the metabolism ofcarbon compounds such as sugars or in the generation of energy moleculesby processes such as oxidative phosphorylation in Corynebacteriumglutamicum. Given the availability of cloning vectors for use inCorynebacterium glutamicum, such as those disclosed in Sinskey et al.,U.S. Pat. No. 4,649,119, and techniques for genetic manipulation of C.glutamicum and the related Brevibacterium species (e.g., lactofermentum)(Yoshihama et al, J. Bacteriol. 162: 591-597 (1985); Katsumata et al.,J. Bacteriol. 159: 306-311 (1984); and Santamaria et al., J. Gen.Microbiol. 130: 2237-2246 (1984)), the nucleic acid molecules of theinvention may be utilized in the genetic engineering of this organism tomake it a better or more efficient producer of one or more finechemicals. This improved production or efficiency of production of afine chemical may be due to a direct effect of manipulation of a gene ofthe invention, or it may be due to an indirect effect of suchmanipulation.

There are a number of mechanisms by which the alteration of an SMPprotein of the invention may directly affect the yield, production,and/or efficiency of production of a fine chemical from a C. glutamicumstrain incorporating such an altered protein. The degradation ofhigh-energy carbon molecules such as sugars, and the conversion ofcompounds such as NADH and FADH₂ to compounds containing high energyphosphate bonds via oxidative phosphorylation results in a number ofcompounds which themselves may be desirable fine chemicals, such aspyruvate, ATP, NADH, and a number of intermediate sugar compounds.Further, the energy molecules (such as ATP) and the reducing equivalents(such as NADH or NADPH) produced by these metabolic pathways areutilized in the cell to drive reactions which would otherwise beenergetically unfavorable. Such unfavorable reactions include manybiosynthetic pathways for fine chemicals. By improving the ability ofthe cell to utilize a particular sugar (e.g., by manipulating the genesencoding enzymes involved in the degradation and conversion of thatsugar into energy for the cell), one may increase the amount of energyavailable to permit unfavorable, yet desired metabolic reactions (e.g.,the biosynthesis of a desired fine chemical) to occur.

The mutagenesis of one or more SMP genes of the invention may alsoresult in SMP proteins having altered activities which indirectly impactthe production of one or more desired fine chemicals from C. glutamicum.For example, by increasing the efficiency of utilization of one or moresugars (such that the conversion of the sugar to useful energy moleculesis improved), or by increasing the efficiency of conversion of reducingequivalents to useful energy molecules (e.g., by improving theefficiency of oxidative phosphorylation, or the activity of the ATPsynthase), one can increase the amount of these high-energy compoundsavailable to the cell to drive normally unfavorable metabolic processes.These processes include the construction of cell walls, transcription,translation, and the biosynthesis of compounds necessary for growth anddivision of the cells (e.g., nucleotides, amino acids, vitamins, lipids,etc.) (Lengeler et al. (1999) Biology of Prokaryotes, Thieme Verlag:Stuttgart, p. 88-109; 913-918; 875-899). By improving the growth andmultiplication of these engineered cells, it is possible to increaseboth the viability of the cells in large-scale culture, and also toimprove their rate of division, such that a relatively larger number ofcells can survive in fermentor culture. The yield, production, orefficiency of production may be increased, at least due to the presenceof a greater number of viable cells, each producing the desired finechemical. Also, many of the degradation products produced during sugarmetabolism are utilized by the cell as precursors or intermediates inthe production of other desirable products, such as fine chemicals. So,by increasing the ability of the cell to metabolize sugars, the numberof these degradation products available to the cell for other processesshould also be increased.

The invention provides novel nucleic acid molecules which encodeproteins, referred to herein as SMP proteins, which are capable of, forexample, performing a function involved in the metabolism of carboncompounds such as sugars and the generation of energy molecules byprocesses such as oxidative phosphorylation in Corynebacteriumglutamicum. Nucleic acid molecules encoding an SMP protein are referredto herein as SMP nucleic acid molecules. In a preferred embodiment, theSMP protein participates in the conversion of carbon molecules anddegradation products thereof to energy which is utilized by the cell formetabolic processes. Examples of such proteins include those encoded bythe genes set forth in Table 1.

Accordingly, one aspect of the invention pertains to isolated nucleicacid molecules (e.g., cDNAs, DNAs, or RNAs) comprising a nucleotidesequence encoding an SMP protein or biologically active portionsthereof, as well as nucleic acid fragments suitable as primers orhybridization probes for the detection or amplification of SMP-encodingnucleic acid (e.g., DNA or mRNA). In particularly preferred embodiments,the isolated nucleic acid molecule comprises one of the nucleotidesequences set forth in Appendix A or the coding region or a complementthereof of one of these nucleotide sequences. In other particularlypreferred embodiments, the isolated nucleic acid molecule of theinvention comprises a nucleotide sequence which hybridizes to or is atleast about 50%, preferably at least about 60%, more preferably at leastabout 70%, 80% or 90%, and even more preferably at least about 95%, 96%,97%, 98%, 99% or more homologous to a nucleotide sequence set forth inAppendix A, or a portion thereof. In other preferred embodiments, theisolated nucleic acid molecule encodes one of the amino acid sequencesset forth in Appendix B. The preferred SMP proteins of the presentinvention also preferably possess at least one of the SMP activitiesdescribed herein.

In another embodiment, the isolated nucleic acid molecule encodes aprotein or portion thereof wherein the protein or portion thereofincludes an amino acid sequence which is sufficiently homologous to anamino acid sequence of Appendix B, e.g., sufficiently homologous to anamino acid sequence of Appendix B such that the protein or portionthereof maintains an SMP activity. Preferably, the protein or portionthereof encoded by the nucleic acid molecule maintains the ability toperform a function involved in the metabolism of carbon compounds suchas sugars or the generation of energy molecules (e.g., ATP) by processessuch as oxidative phosphorylation in Corynebacterium glutamicum. In oneembodiment, the protein encoded by the nucleic acid molecule is at leastabout 50%, preferably at least about 60%, and more preferably at leastabout 70%, 80%, or 90% and most preferably at least about 95%, 96%, 97%,98%, or 99% or more homologous to an amino acid sequence of Appendix B(e.g., an entire amino acid sequence selected from those sequences setforth in Appendix B). In another preferred embodiment, the protein is afull length C. glutamicum protein which is substantially homologous toan entire amino acid sequence of Appendix B (encoded by an open readingframe shown in Appendix A).

In another preferred embodiment, the isolated nucleic acid molecule isderived from C. glutamicum and encodes a protein (e.g., an SMP fusionprotein) which includes a biologically active domain which is at leastabout 50% or more homologous to one of the amino acid sequences ofAppendix B and is able to perform a function involved in the metabolismof carbon compounds such as sugars or the generation of energy molecules(e.g., ATP) by processes such as oxidative phosphorylation inCorynebacterium glutamicum, or has one or more of the activities setforth in Table 1, and which also includes heterologous nucleic acidsequences encoding a heterologous polypeptide or regulatory regions.

In another embodiment, the isolated nucleic acid molecule is at least 15nucleotides in length and hybridizes under stringent conditions to anucleic acid molecule comprising a nucleotide sequence of Appendix A.Preferably, the isolated nucleic acid molecule corresponds to anaturally-occurring nucleic acid molecule. More preferably, the isolatednucleic acid encodes a naturally-occurring C. glutamicum SMP protein, ora biologically active portion thereof.

Another aspect of the invention pertains to vectors, e.g., recombinantexpression vectors, containing the nucleic acid molecules of theinvention, and host cells into which such vectors have been introduced.In one embodiment, such a host cell is used to produce an SMP protein byculturing the host cell in a suitable medium. The SMP protein can bethen isolated from the medium or the host cell.

Yet another aspect of the invention pertains to a genetically alteredmicroorganism in which an SMP gene has been introduced or altered. Inone embodiment, the genome of the microorganism has been altered byintroduction of a nucleic acid molecule of the invention encodingwild-type or mutated SMP sequence as a transgene. In another embodiment,an endogenous SMP gene within the genome of the microorganism has beenaltered, e.g., functionally disrupted, by homologous recombination withan altered SMP gene. In another embodiment, an endogenous or introducedSMP gene in a microorganism has been altered by one or more pointmutations, deletions, or inversions, but still encodes a functional SMPprotein. In still another embodiment, one or more of the regulatoryregions (e.g., a promoter, repressor, or inducer) of an SMP gene in amicroorganism has been altered (e.g., by deletion, truncation,inversion, or point mutation) such that the expression of the SMP geneis modulated. In a preferred embodiment, the microorganism belongs tothe genus Corynebacterium or Brevibacterium, with Corynebacteriumglutamicum being particularly preferred. In a preferred embodiment, themicroorganism is also utilized for the production of a desired compound,such as an amino acid, with lysine being particularly preferred.

In another aspect, the invention provides a method of identifying thepresence or activity of Cornyebacterium diphtheriae in a subject. Thismethod includes detection of one or more of the nucleic acid or aminoacid sequences of the invention (e.g., the sequences set forth inAppendix A or Appendix B) in a subject, thereby detecting the presenceor activity of Corynebacterium diphtheriae in the subject.

Still another aspect of the invention pertains to an isolated SMPprotein or a portion, e.g., a biologically active portion, thereof. In apreferred embodiment, the isolated SMP protein or portion thereof iscapable of performing a function involved in the metabolism of carboncompounds such as sugars or in the generation of energy molecules (e.g.,ATP) by processes such as oxidative phosphorylation in Corynebacteriumglutamicum. In another preferred embodiment, the isolated SMP protein orportion thereof is sufficiently homologous to an amino acid sequence ofAppendix B such that the protein or portion thereof maintains theability to perform a function involved in the metabolism of carboncompounds such as sugars or in the generation of energy molecules (e.g.,ATP) by processes such as oxidative phosphorylation in Corynebacteriumglutamicum.

The invention also provides an isolated preparation of an SMP protein.In preferred embodiments, the SMP protein comprises an amino acidsequence of Appendix B. In another preferred embodiment, the inventionpertains to an isolated full length protein which is substantiallyhomologous to an entire amino acid sequence of Appendix B (encoded by anopen reading frame set forth in Appendix A). In yet another embodiment,the protein is at least about 50%, preferably at least about 60%, andmore preferably at least about 70%, 80%, or 90%, and most preferably atleast about 95%, 96%, 97%, 98%, or 99% or more homologous to an entireamino acid sequence of Appendix B. In other embodiments, the isolatedSMP protein comprises an amino acid sequence which is at least about 50%or more homologous to one of the amino acid sequences of Appendix B andis able to perform a function involved in the metabolism of carboncompounds such as sugars or in the generation of energy molecules (e.g.,ATP) by processes such as oxidative phosphorylation in Corynebacteriumglutamicum, or has one or more of the activities set forth in Table 1.

Alternatively, the isolated SMP protein can comprise an amino acidsequence which is encoded by a nucleotide sequence which hybridizes,e.g., hybridizes under stringent conditions, or is at least about 50%,preferably at least about 60%, more preferably at least about 70%, 80%,or 90%, and even more preferably at least about 95%, 96%, 97%, 98,%, or99% or more homologous, to a nucleotide sequence of Appendix B. It isalso preferred that the preferred forms of SMP proteins also have one ormore of the SMP bioactivities described herein.

The SMP polypeptide, or a biologically active portion thereof, can beoperatively linked to a non-SMP polypeptide to form a fusion protein. Inpreferred embodiments, this fusion protein has an activity which differsfrom that of the SMP protein alone. In other preferred embodiments, thisfusion protein performs a function involved in the metabolism of carboncompounds such as sugars or in the generation of energy molecules (e.g.,ATP) by processes such as oxidative phosphorylation in Corynebacteriumglutamicum. In particularly preferred embodiments, integration of thisfusion protein into a host cell modulates production of a desiredcompound from the cell.

In another aspect, the invention provides methods for screeningmolecules which modulate the activity of an SMP protein, either byinteracting with the protein itself or a substrate or binding partner ofthe SMP protein, or by modulating the transcription or translation of anSMP nucleic acid molecule of the invention.

Another aspect of the invention pertains to a method for producing afine chemical. This method involves the culturing of a cell containing avector directing the expression of an SMP nucleic acid molecule of theinvention, such that a fine chemical is produced. In a preferredembodiment, this method further includes the step of obtaining a cellcontaining such a vector, in which a cell is transfected with a vectordirecting the expression of an SMP nucleic acid. In another preferredembodiment, this method further includes the step of recovering the finechemical from the culture. In a particularly preferred embodiment, thecell is from the genus Corynebacterium or Brevibacterium, or is selectedfrom those strains set forth in Table 3.

Another aspect of the invention pertains to methods for modulatingproduction of a molecule from a microorganism. Such methods includecontacting the cell with an agent which modulates SMP protein activityor SMP nucleic acid expression such that a cell associated activity isaltered relative to this same activity in the absence of the agent. In apreferred embodiment, the cell is modulated for one or more C.glutamicum carbon metabolism pathways or for the production of energythrough processes such as oxidative phosphorylation, such that theyields or rate of production of a desired fine chemical by thismicroorganism is improved. The agent which modulates SMP proteinactivity can be an agent which stimulates SMP protein activity or SMPnucleic acid expression. Examples of agents which stimulate SMP proteinactivity or SMP nucleic acid expression include small molecules, activeSMP proteins, and nucleic acids encoding SMP proteins that have beenintroduced into the cell. Examples of agents which inhibit SMP activityor expression include small molecules and antisense SMP nucleic acidmolecules.

Another aspect of the invention pertains to methods for modulatingyields of a desired compound from a cell, involving the introduction ofa wild-type or mutant SMP gene into a cell, either maintained on aseparate plasmid or integrated into the genome of the host cell. Ifintegrated into the genome, such integration can be random, or it cantake place by homologous recombination such that the native gene isreplaced by the introduced copy, causing the production of the desiredcompound from the cell to be modulated. In a preferred embodiment, saidyields are increased. In another preferred embodiment, said chemical isa fine chemical. In a particularly preferred embodiment, said finechemical is an amino acid. In especially preferred embodiments, saidamino acid is L-lysine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides SMP nucleic acid and protein moleculeswhich are involved in the metabolism of carbon compounds such as sugarsand the generation of energy molecules by processes such as oxidativephosphorylation in Corynebacterium glutamicum. The molecules of theinvention may be utilized in the modulation of production of finechemicals from microorganisms, such as C. glutamicum, either directly(e.g., where overexpression or optimization of a glycolytic pathwayprotein has a direct impact on the yield, production, and/or efficiencyof production of, e.g., pyruvate from modified C. glutamicum), or mayhave an indirect impact which nonetheless results in an increase ofyield, production, and/or efficiency of production of the desiredcompound (e.g., where modulation of proteins involved in oxidativephosphorylation results in alterations in the amount of energy availableto perform necessary metabolic processes and other cellular functions,such as nucleic acid and protein biosynthesis andtranscription/translation). Aspects of the invention are furtherexplicated below.

I. Fine Chemicals

The term ‘fine chemical’ is art-recognized and includes moleculesproduced by an organism which have applications in various industries,such as, but not limited to, the pharmaceutical, agriculture, andcosmetics industries. Such compounds include organic acids, such astartaric acid, itaconic acid, and diaminopimelic acid, bothproteinogenic and non-proteinogenic amino acids, purine and pyrimidinebases, nucleosides, and nucleotides (as described e.g. in Kuninaka, A.(1996) Nucleotides and related compounds, p. 561-612, in Biotechnologyvol. 6, Rehm et al., eds. VCH: Weinheim, and references containedtherein), lipids, both saturated and unsaturated fatty acids (e.g.,arachidonic acid), diols (e.g., propane diol, and butane diol),carbohydrates (e.g., hyaluronic acid and trehalose), aromatic compounds(e.g., aromatic amines, vanillin, and indigo), vitamins and cofactors(as described in Ullmann's Encyclopedia of Industrial Chemistry, vol.A27, “Vitamins”, p. 443-613 (1996) VCH: Weinheim and references therein;and Ong, A. S., Niki, E. & Packer, L. (1995) “Nutrition, Lipids, Health,and Disease” Proceedings of the UNESCO/Confederation of Scientific andTechnological Associations in Malaysia, and the Society for Free RadicalResearch Asia, held Sep. 1-3, 1994 at Penang, Malaysia, AOCS Press,(1995)), enzymes, polyketides (Cane et al. (1998) Science 282: 63-68),and all other chemicals described in Gutcho (1983) Chemicals byFermentation, Noyes Data Corporation, ISBN: 0818805086 and referencestherein. The metabolism and uses of certain of these fine chemicals arefurther explicated below.

A. Amino Acid Metabolism and Uses

Amino acids comprise the basic structural units of all proteins, and assuch are essential for normal cellular functioning in all organisms. Theterm “amino acid” is art-recognized. The proteinogenic amino acids, ofwhich there are 20 species, serve as structural units for proteins, inwhich they are linked by peptide bonds, while the nonproteinogenic aminoacids (hundreds of which are known) are not normally found in proteins(see Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97VCH: Weinheim (1985)). Amino acids may be in the D- or L-opticalconfiguration, though L-amino acids are generally the only type found innaturally-occurring proteins. Biosynthetic and degradative pathways ofeach of the 20 proteinogenic amino acids have been well characterized inboth prokaryotic and eukaryotic cells (see, for example, Stryer, L.Biochemistry, 3^(rd) edition, pages 578-590 (1988)). The ‘essential’amino acids (histidine, isoleucine, leucine, lysine, methionine,phenylalanine, threonine, tryptophan, and valine), so named because theyare generally a nutritional requirement due to the complexity of theirbiosyntheses, are readily converted by simple biosynthetic pathways tothe remaining 11 ‘nonessential’ amino acids (alanine, arginine,asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline,serine, and tyrosine). Higher animals do retain the ability tosynthesize some of these amino acids, but the essential amino acids mustbe supplied from the diet in order for normal protein synthesis tooccur.

Aside from their function in protein biosynthesis, these amino acids areinteresting chemicals in their own right, and many have been found tohave various applications in the food, feed, chemical, cosmetics,agriculture, and pharmaceutical industries. Lysine is an important aminoacid in the nutrition not only of humans, but also of monogastricanimals such as poultry and swine. Glutamate is most commonly used as aflavor additive (mono-sodium glutamate, MSG) and is widely usedthroughout the food industry, as are aspartate, phenylalanine, glycine,and cysteine. Glycine, L-methionine and tryptophan are all utilized inthe pharmaceutical industry. Glutamine, valine, leucine, isoleucine,histidine, arginine, proline, serine and alanine are of use in both thepharmaceutical and cosmetics industries. Threonine, tryptophan, andD/L-methionine are common feed additives. (Leuchtenberger, W. (1996)Amino aids—technical production and use, p. 466-502 in Rehm et al.(eds.) Biotechnology vol. 6, chapter 14a, VCH: Weinheim). Additionally,these amino acids have been found to be useful as precursors for thesynthesis of synthetic amino acids and proteins, such asN-acetylcysteine, S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan,and others described in Ulmann's Encyclopedia of Industrial Chemistry,vol. A2, p. 57-97, VCH: Weinheim, 1985.

The biosynthesis of these natural amino acids in organisms capable ofproducing them, such as bacteria, has been well characterized (forreview of bacterial amino acid biosynthesis and regulation thereof, seeUmbarger, H. E. (1978) Ann. Rev. Biochem. 47: 533-606). Glutamate issynthesized by the reductive amination of α-ketoglutarate, anintermediate in the citric acid cycle. Glutamine, proline, and arginineare each subsequently produced from glutamate. The biosynthesis ofserine is a three-step process beginning with 3-phosphoglycerate (anintermediate in glycolysis), and resulting in this amino acid afteroxidation, transamination, and hydrolysis steps. Both cysteine andglycine are produced from serine; the former by the condensation ofhomocysteine with serine, and the latter by the transferal of theside-chain β-carbon atom to tetrahydrofolate, in a reaction catalyzed byserine transhydroxymethylase. Phenylalanine, and tyrosine aresynthesized from the glycolytic and pentose phosphate pathway precursorserythrose 4-phosphate and phosphoenolpyruvate in a 9-step biosyntheticpathway that differ only at the final two steps after synthesis ofprephenate. Tryptophan is also produced from these two initialmolecules, but its synthesis is an 11-step pathway. Tyrosine may also besynthesized from phenylalanine, in a reaction catalyzed by phenylalaninehydroxylase. Alanine, valine, and leucine are all biosynthetic productsof pyruvate, the final product of glycolysis. Aspartate is formed fromoxaloacetate, an intermediate of the citric acid cycle. Asparagine,methionine, threonine, and lysine are each produced by the conversion ofaspartate. Isoleucine is formed from threonine. A complex 9-step pathwayresults in the production of histidine from5-phosphoribosyl-1-pyrophosphate, an activated sugar.

Amino acids in excess of the protein synthesis needs of the cell cannotbe stored, and are instead degraded to provide intermediates for themajor metabolic pathways of the cell (for review see Stryer, L.Biochemistry 3^(rd) ed. Ch. 21 “Amino Acid Degradation and the UreaCycle” p. 495-516 (1988)). Although the cell is able to convert unwantedamino acids into useful metabolic intermediates, amino acid productionis costly in terms of energy, precursor molecules, and the enzymesnecessary to synthesize them. Thus it is not surprising that amino acidbiosynthesis is regulated by feedback inhibition, in which the presenceof a particular amino acid serves to slow or entirely stop its ownproduction (for overview of feedback mechanisms in amino acidbiosynthetic pathways, see Stryer, L. Biochemistry, 3^(rd) ed. Ch. 24:“Biosynthesis of Amino Acids and Heme” p. 575-600 (1988)). Thus, theoutput of any particular amino acid is limited by the amount of thatamino acid present in the cell.

B. Vitamin, Cofactor, and Nutraceutical Metabolism and Uses

Vitamins, cofactors, and nutraceuticals comprise another group ofmolecules which the higher animals have lost the ability to synthesizeand so must ingest, although they are readily synthesized by otherorganisms such as bacteria. These molecules are either bioactivesubstances themselves, or are precursors of biologically activesubstances which may serve as electron carriers or intermediates in avariety of metabolic pathways. Aside from their nutritive value, thesecompounds also have significant industrial value as coloring agents,antioxidants, and catalysts or other processing aids. (For an overviewof the structure, activity, and industrial applications of thesecompounds, see, for example, Ullman's Encyclopedia of IndustrialChemistry, “Vitamins” vol. A27, p. 443-613, VCH: Weinheim, 1996.) Theterm “vitamin” is art-recognized, and includes nutrients which arerequired by an organism for normal functioning, but which that organismcannot synthesize by itself. The group of vitamins may encompasscofactors and nutraceutical compounds. The language “cofactor” includesnonproteinaceous compounds required for a normal enzymatic activity tooccur. Such compounds may be organic or inorganic; the cofactormolecules of the invention are preferably organic. The term“nutraceutical” includes dietary supplements having health benefits inplants and animals, particularly humans. Examples of such molecules arevitamins, antioxidants, and also certain lipids (e.g., polyunsaturatedfatty acids).

The biosynthesis of these molecules in organisms capable of producingthem, such as bacteria, has been largely characterized (Ullman'sEncyclopedia of Industrial Chemistry, “Vitamins” vol. A27, p. 443-613,VCH: Weinheim, 1996; Michal, G. (1999) Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley & Sons; Ong, A. S., Niki,E. & Packer, L. (1995) “Nutrition, Lipids, Health, and Disease”Proceedings of the UNESCO/Confederation of Scientific and TechnologicalAssociations in Malaysia, and the Society for Free RadicalResearch—Asia, held Sep. 1-3, 1994 at Penang, Malaysia, AOCS Press:Champaign, Ill. X, 374 S).

Thiamin (vitamin B₁) is produced by the chemical coupling of pyrimidineand thiazole moieties. Riboflavin (vitamin B₂) is synthesized fromguanosine-5′-triphosphate (GTP) and ribose-5′-phosphate. Riboflavin, inturn, is utilized for the synthesis of flavin mononucleotide (FMN) andflavin adenine dinucleotide (FAD). The family of compounds collectivelytermed ‘vitamin B₆’ (e.g., pyridoxine, pyridoxamine,pyridoxa-5′-phosphate, and the commercially used pyridoxinhydrochloride) are all derivatives of the common structural unit,5-hydroxy-6-methylpyridine. Pantothenate (pantothenic acid,(R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-β-alanine) can beproduced either by chemical synthesis or by fermentation. The finalsteps in pantothenate biosynthesis consist of the ATP-drivencondensation of β-alanine and pantoic acid. The enzymes responsible forthe biosynthesis steps for the conversion to pantoic acid, to β-alanineand for the condensation to panthotenic acid are known. Themetabolically active form of pantothenate is Coenzyme A, for which thebiosynthesis proceeds in 5 enzymatic steps. Pantothenate,pyridoxal-5′-phosphate, cysteine and ATP are the precursors of CoenzymeA. These enzymes not only catalyze the formation of panthothante, butalso the production of (R)-pantoic acid, (R)-pantolacton, (R)-panthenol(provitamin B₅), pantetheine (and its derivatives) and coenzyme A.

Biotin biosynthesis from the precursor molecule pimeloyl-CoA inmicroorganisms has been studied in detail and several of the genesinvolved have been identified. Many of the corresponding proteins havebeen found to also be involved in Fe-cluster synthesis and are membersof the nifS class of proteins. Lipoic acid is derived from octanoicacid, and serves as a coenzyme in energy metabolism, where it becomespart of the pyruvate dehydrogenase complex and the α-ketoglutaratedehydrogenase complex. The folates are a group of substances which areall derivatives of folic acid, which is turn is derived from L-glutamicacid, p-amino-benzoic acid and 6-methylpterin. The biosynthesis of folicacid and its derivatives, starting from the metabolism intermediatesguanosine-5′-triphosphate (GTP), L-glutamic acid and p-amino-benzoicacid has been studied in detail in certain microorganisms.

Corrinoids (such as the cobalamines and particularly vitamin B₁₂) andporphyrines belong to a group of chemicals characterized by atetrapyrole ring system. The biosynthesis of vitamin B₁₂ is sufficientlycomplex that it has not yet been completely characterized, but many ofthe enzymes and substrates involved are now known. Nicotinic acid(nicotinate), and nicotinamide are pyridine derivatives which are alsotermed ‘niacin’. Niacin is the precursor of the important coenzymes NAD(nicotinamide adenine dinucleotide) and NADP (nicotinamide adeninedinucleotide phosphate) and their reduced forms.

The large-scale production of these compounds has largely relied oncell-free chemical syntheses, though some of these chemicals have alsobeen produced by large-scale culture of microorganisms, such asriboflavin, Vitamin B₆, pantothenate, and biotin. Only Vitamin B₁₂ isproduced solely by fermentation, due to the complexity of its synthesis.In vitro methodologies require significant inputs of materials and time,often at great cost.

C. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and Uses

Purine and pyrimidine metabolism genes and their corresponding proteinsare important targets for the therapy of tumor diseases and viralinfections. The language “purine” or “pyrimidine” includes thenitrogenous bases which are constituents of nucleic acids, co-enzymes,and nucleotides. The term “nucleotide” includes the basic structuralunits of nucleic acid molecules, which are comprised of a nitrogenousbase, a pentose sugar (in the case of RNA, the sugar is ribose; in thecase of DNA, the sugar is D-deoxyribose), and phosphoric acid. Thelanguage “nucleoside” includes molecules which serve as precursors tonucleotides, but which are lacking the phosphoric acid moiety thatnucleotides possess. By inhibiting the biosynthesis of these molecules,or their mobilization to form nucleic acid molecules, it is possible toinhibit RNA and DNA synthesis; by inhibiting this activity in a fashiontargeted to cancerous cells, the ability of tumor cells to divide andreplicate may be inhibited. Additionally, there are nucleotides which donot form nucleic acid molecules, but rather serve as energy stores(i.e., AMP) or as coenzymes (i.e., FAD and NAD).

Several publications have described the use of these chemicals for thesemedical indications, by influencing purine and/or pyrimidine metabolism(e.g. Christopherson, R. I. and Lyons, S. D. (1990) “Potent inhibitorsof de novo pyrimidine and purine biosynthesis as chemotherapeuticagents.” Med. Res. Reviews 10: 505-548). Studies of enzymes involved inpurine and pyrimidine metabolism have been focused on the development ofnew drugs which can be used, for example, as immunosuppressants oranti-proliferants (Smith, J. L., (1995) “Enzymes in nucleotidesynthesis.” Curr. Opin. Struct. Biol. 5: 752-757; (1995) Biochem Soc.Transact. 23: 877-902). However, purine and pyrimidine bases,nucleosides and nucleotides have other utilities: as intermediates inthe biosynthesis of several fine chemicals (e.g., thiamine,S-adenosyl-methionine, folates, or riboflavin), as energy carriers forthe cell (e.g., ATP or GTP), and for chemicals themselves, commonly usedas flavor enhancers (e.g., IMP or GMP) or for several medicinalapplications (see, for example, Kuninaka, A. (1996) Nucleotides andRelated Compounds in Biotechnology vol. 6, Rehm et al., eds. VCH:Weinheim, p. 561-612). Also, enzymes involved in purine, pyrimidine,nucleoside, or nucleotide metabolism are increasingly serving as targetsagainst which chemicals for crop protection, including fungicides,herbicides and insecticides, are developed.

The metabolism of these compounds in bacteria has been characterized(for reviews see, for example, Zalkin, H. and Dixon, J. E. (1992) “denovo purine nucleotide biosynthesis”, in: Progress in Nucleic AcidResearch and Molecular Biology, vol. 42, Academic Press:, p. 259-287;and Michal, G. (1999) “Nucleotides and Nucleosides”, Chapter 8 in:Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology,Wiley: New York). Purine metabolism has been the subject of intensiveresearch, and is essential to the normal functioning of the cell.Impaired purine metabolism in higher animals can cause severe disease,such as gout. Purine nucleotides are synthesized fromribose-5-phosphate, in a series of steps through the intermediatecompound inosine-5′-phosphate (IMP), resulting in the production ofguanosine-5′-monophosphate (GMP) or adenosine-5′-monophosphate (AMP),from which the triphosphate forms utilized as nucleotides are readilyformed. These compounds are also utilized as energy stores, so theirdegradation provides energy for many different biochemical processes inthe cell. Pyrimidine biosynthesis proceeds by the formation ofuridine-5′-monophosphate (UMP) from ribose-5-phosphate. UMP, in turn, isconverted to cytidine-5′-triphosphate (CTP). The deoxy-forms of all ofthese nucleotides are produced in a one step reduction reaction from thediphosphate ribose form of the nucleotide to the diphosphate deoxyriboseform of the nucleotide. Upon phosphorylation, these molecules are ableto participate in DNA synthesis.

D. Trehalose Metabolism and Uses

Trehalose consists of two glucose molecules, bound in α,α-1,1 linkage.It is commonly used in the food industry as a sweetener, an additive fordried or frozen foods, and in beverages. However, it also hasapplications in the pharmaceutical, cosmetics and biotechnologyindustries (see, for example, Nishimoto et al., (1998) U.S. Pat. No.5,759,610; Singer, M. A. and Lindquist, S. (1998) Trends Biotech. 16:460-467; Paiva, C. L. A. and Panek, A. D. (1996) Biotech. Ann. Rev. 2:293-314; and Shiosaka, M. (1997) J. Japan 172: 97-102). Trehalose isproduced by enzymes from many microorganisms and is naturally releasedinto the surrounding medium, from which it can be collected usingmethods known in the art.

II. Sugar and Carbon Molecule Utilization and Oxidative Phosphorylation

Carbon is a critically important element for the formation of allorganic compounds, and thus is a nutritional requirement not only forthe growth and division of C. glutamicum, but also for theoverproduction of fine chemicals from this microorganism. Sugars, suchas mono-, di-, or polysaccharides, are particularly good carbon sources,and thus standard growth media typically contain one or more of:glucose, fructose, mannose, galactose, ribose, sorbose, ribulose,lactose, maltose, sucrose, raffinose, starch, or cellulose (Ullmann'sEncyclopedia of Industrial Chemistry (1987) vol. A9, “Enzymes”, VCH:Weinheim). Alternatively, more complex forms of sugar may be utilized inthe media, such as molasses, or other by-products of sugar refinement.Other compounds aside from the sugars may be used as alternate carbonsources, including alcohols (e.g., ethanol or methanol), alkanes, sugaralcohols, fatty acids, and organic acids (e.g., acetic acid or lacticacid). For a review of carbon sources and their utilization bymicroorganisms in culture, see: Ullman's Encyclopedia of IndustrialChemistry (1987) vol. A9, “Enzymes”, VCH: Weinheim; Stoppok, E. andBuchholz, K. (1996) “Sugar-based raw materials for fermentationapplications” in Biotechnology (Rehm, H. J. et al., eds.) vol. 6, VCH:Weinheim, p. 5-29; Rehm, H. J. (1980) Industrielle Mikrobiologie,Springer: Berlin; Bartholomew, W. H., and Reiman, H. B. (1979).Economics of Fermentation Processes, in: Peppler, H. J. and Perlman, D.,eds. Microbial Technology 2^(nd) ed., vol. 2, chapter 18, AcademicPress: New York; and Kockova-Kratachvilova, A. (1981) Characteristics ofIndustrial Microorganisms, in: Rehm, H. J. and Reed, G., eds. Handbookof Biotechnology, vol. 1, chapter 1, Verlag Chemie: Weinheim.

After uptake, these energy-rich carbon molecules must be processed suchthat they are able to be degraded by one of the major sugar metabolicpathways. Such pathways lead directly to useful degradation products,such as ribose-5-phosphate and phosphoenolpyruvate, which may besubsequently converted to pyruvate. Three of the most important pathwaysin bacteria for sugar metabolism include the Embden-Meyerhoff-Pamas(EMP) pathway (also known as the glycolytic or fructose bisphosphatepathway), the hexosemonophosphate (HMP) pathway (also known as thepentose shunt or pentose phosphate pathway), and the Entner-Doudoroff(ED) pathway (for review, see Michal, G. (1999) Biochemical Pathways: AnAtlas of Biochemistry and Molecular Biology, Wiley: New York, andStryer, L. (1988) Biochemistry, Chapters 13-19, Freeman: New York, andreferences therein).

The EMP pathway converts hexose molecules to pyruvate, and in theprocess produces 2 molecules of ATP and 2 molecules of NADH. Startingwith glucose-1-phosphate (which may be either directly taken up from themedium, or alternatively may be generated from glycogen, starch, orcellulose), the glucose molecule is isomerized to fructose-6-phosphate,is phosphorylated, and split into two 3-carbon molecules ofglyceraldehyde-3-phosphate. After dehydrogenation, phosphorylation, andsuccessive rearrangements, pyruvate results.

The HMP pathway converts glucose to reducing equivalents, such as NADPH,and produces pentose and tetrose compounds which are necessary asintermediates and precursors in a number of other metabolic pathways. Inthe HMP pathway, glucose-6-phosphate is converted toribulose-5-phosphate by two successive dehydrogenase reactions (whichalso release two NADPH molecules), and a carboxylation step.Ribulose-5-phosphate may also be converted to xyulose-5-phosphate andribose-5-phosphate; the former can undergo a series of biochemical stepsto glucose-6-phosphate, which may enter the EMP pathway, while thelatter is commonly utilized as an intermediate in other biosyntheticpathways within the cell.

The ED pathway begins with the compound glucose or gluconate, which issubsequently phosphorylated and dehydrated to form2-dehydro-3-deoxy-6-P-gluconate. Glucuronate and galacturonate may alsobe converted to 2-dehydro-3-deoxy-6-P-gluconate through more complexbiochemical pathways. This product molecule is subsequently cleaved intoglyceraldehyde-3-P and pyruvate; glyceraldehyde-3-P may itself also beconverted to pyruvate.

The EMP and HMP pathways share many features, including intermediatesand enzymes. The EMP pathway provides the greatest amount of ATP, but itdoes not produce ribose-5-phosphate, an important precursor for, e.g.,nucleic acid biosynthesis, nor does it produce erythrose-4-phosphate,which is important for amino acid biosynthesis. Microorganisms that arecapable of using only the EMP pathway for glucose utilization are thusnot able to grow on simple media with glucose as the sole carbon source.They are referred to as fastidious organisms, and their growth requiresinputs of complex organic compounds, such as those found in yeastextract.

In contrast, the HMP pathway produces all of the precursors necessaryfor both nucleic acid and amino acid biosynthesis, yet yields only halfthe amount of ATP energy that the EMP pathway does. The HMP pathway alsoproduces NADPH, which may be used for redox reactions in biosyntheticpathways. The HMP pathway does not directly produce pyruvate, however,and thus these microorganisms must also possess this portion of the EMPpathway. It is therefore not surprising that a number of microorganisms,particularly the facultative anerobes, have evolved such that theypossess both of these pathways.

The ED pathway has thus far has only been found in bacteria. Althoughthis pathway is linked partly to the HMP pathway in the reversedirection for precursor formation, the ED pathway directly formspyruvate by the aldolase cleavage of 3-ketodeoxy-6-phosphogluconate. TheED pathway can exist on its own and is utilized by the majority ofstrictly aerobic microorganisms. The net result is similar to that ofthe HMP pathway, although one mole of ATP can be formed only if thecarbon atoms are converted into pyruvate, instead of into precursormolecules.

The pyruvate molecules produced through any of these pathways can bereadily converted into energy via the Krebs cycle (also known as thecitric acid cycle, the citrate cycle, or the tricarboxylic acid cycle(TCA cycle)). In this process, pyruvate is first decarboxylated,resulting in the production of one molecule of NADH, 1 molecule ofacetyl-CoA, and 1 molecule of CO₂. The acetyl group of acetyl CoA thenreacts with the 4 carbon unit, oxaolacetate, leading to the formation ofcitric acid, a 6 carbon organic acid. Dehydration and two additional CO₂molecules are released. Ultimately, oxaloacetate is regenerated and canserve again as an acetyl acceptor, thus completing the cycle. Theelectrons released during the oxidation of intermediates in the TCAcycle are transferred to NAD³⁰ to yield NADH.

During respiration, the electrons from NADH are transferred to molecularoxygen or other terminal electron acceptors. This process is catalyzedby the respiratory chain, an electron transport system containing bothintegral membrane proteins and membrane associated proteins. This systemserves two basic functions: first, to accept electrons from an electrondonor and to transfer them to an electron acceptor, and second, toconserve some of the energy released during electron transfer by thesynthesis of ATP. Several types of oxidation-reduction enzymes andelectron transport proteins are known to be involved in such processes,including the NADH dehydrogenases, flavin-containing electron carriers,iron sulfur proteins, and cytochromes. The NADH dehydrogenases arelocated at the cytoplasmic surface of the plasma membrane, and transferhydrogen atoms from NADH to flavoproteins, in turn accepting electronsfrom NADH. The flavoproteins are a group of electron carriers possessinga flavin prosthetic group which is alternately reduced and oxidized asit accepts and transfers electrons Three flavins are known toparticipate in these reactions: riboflavin, flavin-adenine dinucleotide(FAD) and flavin-mononucleotide (FMN). Iron sulfur proteins contain acluster of iron and sulfur atoms which are not bonded to a heme group,but which still are able to participate in dehydration and rehydrationreactions. Succinate dehydrogenase and aconitase are exemplaryiron-sulfur proteins; their iron-sulfur complexes serve to accept andtransfer electrons as part of the overall electron-transport chain. Thecytochromes are proteins containing an iron porphyrin ring (heme). Thereare a number of different classes of cytochromes, differing in theirreduction potentials. Functionally, these cytochromes form pathways inwhich electrons may be transferred to other cytochromes havingincreasingly more positive reduction potentials. A further class ofnon-protein electron carriers is known: the lipid-soluble quinones(e.g., coenzyme Q). These molecules also serve as hydrogen atomacceptors and electron donors.

The action of the respiratory chain generates a proton gradient acrossthe cell membrane, resulting in proton motive force. This force isutilized by the cell to synthesize ATP, via the membrane-spanningenzyme, ATP synthase. This enzyme is a multiprotein complex in which thetransport of H⁺ molecules through the membrane results in the physicalrotation of the intracellular subunits and concomitant phosphorylationof ADP to form ATP (for review, see Fillingame, R. H. and Divall, S.(1999) Novartis Found. Symp. 221: 218-229, 229-234).

Non-hexose carbon substrates may also serve as carbon and energy sourcesfor cells. Such substrates may first be converted to hexose sugars inthe gluconeogenesis pathway, where glucose is first synthesized by thecell and then is degraded to produce energy. The starting material forthis reaction is phosphoenolpyruvate (PEP), which is one of the keyintermediates in the glycolytic pathway. PEP may be formed fromsubstrates other than sugars, such as acetic acid, or by decarboxylationof oxaloacetate (itself an intermediate in the TCA cycle). By reversingthe glycolytic pathway (utilizing a cascade of enzymes different thanthose of the original glycolysis pathway), glucose-6-phosphate may beformed. The conversion of pyruvate to glucose requires the utilizationof 6 high energy phosphate bonds, whereas glycolysis only produces 2 ATPin the conversion of glucose to pyruvate. However, the completeoxidation of glucose (glycolysis, conversion of pyruvate into acetylCoA, citric acid cycle, and oxidative phosphorylation) yields between36-38 ATP, so the net loss of high energy phosphate bonds experiencedduring gluconeogenesis is offset by the overall greater gain in suchhigh-energy molecules produced by the oxidation of glucose.

III. Elements and Methods of the Invention

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as SMP nucleic acid and proteinmolecules, which participate in the conversion of sugars to usefuldegradation products and energy (e.g., ATP) in C. glutamicum or whichmay participate in the production of useful energy-rich molecules (e.g.,ATP) by other processes, such as oxidative phosphorylation. In oneembodiment, the SMP molecules participate in the metabolism of carboncompounds such as sugars or the generation of energy molecules (e.g.,ATP) by processes such as oxidative phosphorylation in Corynebacteriumglutamicum. In a preferred embodiment, the activity of the SMP moleculesof the present invention to contribute to carbon metabolism or energyproduction in C. glutamicum has an impact on the production of a desiredfine chemical by this organism. In a particularly preferred embodiment,the SMP molecules of the invention are modulated in activity, such thatthe C. glutamicum metabolic and energetic pathways in which the SMPproteins of the invention participate are modulated in yield,production, and/or efficiency of production, which either directly orindirectly modulates the yield, production, and/or efficiency ofproduction of a desired fine chemical by C. glutamicum.

The language, “SMP protein” or “SMP polypeptide” includes proteins whichare capable of performing a function involved in the metabolism ofcarbon compounds such as sugars and the generation of energy moleculesby processes such as oxidative phosphorylation in Corynebacteriumglutamicum. Examples of SMP proteins include those encoded by the SMPgenes set forth in Table 1 and Appendix A. The terms “SMP gene” or “SMPnucleic acid sequence” include nucleic acid sequences encoding an SMPprotein, which consist of a coding region and also correspondinguntranslated 5′ and 3′ sequence regions. Examples of SMP genes includethose set forth in Table 1. The terms “production” or “productivity” areart-recognized and include the concentration of the fermentation product(for example, the desired fine chemical) formed within a given time anda given fermentation volume (e.g., kg product per hour per liter). Theterm “efficiency of production” includes the time required for aparticular level of production to be achieved (for example, how long ittakes for the cell to attain a particular rate of output of a finechemical). The term “yield” or “product/carbon yield” is art-recognizedand includes the efficiency of the conversion of the carbon source intothe product (i.e., fine chemical). This is generally written as, forexample, kg product per kg carbon source. By increasing the yield orproduction of the compound, the quantity of recovered molecules, or ofuseful recovered molecules of that compound in a given amount of cultureover a given amount of time is increased. The terms “biosynthesis” or a“biosynthetic pathway” are art-recognized and include the synthesis of acompound, preferably an organic compound, by a cell from intermediatecompounds in what may be a multistep and highly regulated process. Theterms “degradation” or a “degradation pathway” are art-recognized andinclude the breakdown of a compound, preferably an organic compound, bya cell to degradation products (generally speaking, smaller or lesscomplex molecules) in what may be a multistep and highly regulatedprocess. The term “degradation product” is art-recognized and includesbreakdown products of a compound. Such products may themselves haveutility as precursor (starting point) or intermediate moleculesnecessary for the biosynthesis of other compounds by the cell. Thelanguage “metabolism” is art-recognized and includes the totality of thebiochemical reactions that take place in an organism. The metabolism ofa particular compound, then, (e.g., the metabolism of an amino acid suchas glycine) comprises the overall biosynthetic, modification, anddegradation pathways in the cell related to this compound.

In another embodiment, the SMP molecules of the invention are capable ofmodulating the production of a desired molecule, such as a finechemical, in a microorganism such as C. glutamicum. There are a numberof mechanisms by which the alteration of an SMP protein of the inventionmay directly affect the yield, production, and/or efficiency ofproduction of a fine chemical from a C. glutamicum strain incorporatingsuch an altered protein. The degradation of high-energy carbon moleculessuch as sugars, and the conversion of compounds such as NADH and FADH₂to more useful forms via oxidative phosphorylation results in a numberof compounds which themselves may be desirable fine chemicals, such aspyruvate, ATP, NADH, and a number of intermediate sugar compounds.Further, the energy molecules (such as ATP) and the reducing equivalents(such as NADH or NADPH) produced by these metabolic pathways areutilized in the cell to drive reactions which would otherwise beenergetically unfavorable. Such unfavorable reactions include manybiosynthetic pathways for fine chemicals. By improving the ability ofthe cell to utilize a particular sugar (e.g., by manipulating the genesencoding enzymes involved in the degradation and conversion of thatsugar into energy for the cell), one may increase the amount of energyavailable to permit unfavorable, yet desired metabolic reactions (e.g.,the biosynthesis of a desired fine chemical) to occur.

The mutagenesis of one or more SMP genes of the invention may alsoresult in SMP proteins having altered activities which indirectly impactthe production of one or more desired fine chemicals from C. glutamicum.For example, by increasing the efficiency of utilization of one or moresugars (such that the conversion of the sugar to useful energy moleculesis improved), or by increasing the efficiency of conversion of reducingequivalents to useful energy molecules (e.g., by improving theefficiency of oxidative phosphorylation, or the activity of the ATPsynthase), one can increase the amount of these high-energy compoundsavailable to the cell to drive normally unfavorable metabolic processes.These processes include the construction of cell walls, transcription,translation, and the biosynthesis of compounds necessary for growth anddivision of the cells (e.g., nucleotides, amino acids, vitamins, lipids,etc.) (Lengeler et al. (1999) Biology of Prokaryotes, Thieme Verlag:Stuttgart, p. 88-109; 913-918; 875-899). By improving the growth andmultiplication of these engineered cells, it is possible to increaseboth the viability of the cells in large-scale culture, and also toimprove their rate of division, such that a relatively larger number ofcells can survive in fermentor culture. The yield, production, orefficiency of production may be increased, at least due to the presenceof a greater number of viable cells, each producing the desired finechemical. Further, a number of the degradation and intermediatecompounds produced during sugar metabolism are necessary precursors andintermediates for other biosynthetic pathways throughout the cell. Forexample, many amino acids are synthesized directly from compoundsnormally resulting from glycolysis or the TCA cycle (e.g., serine issynthesized from 3-phosphoglycerate, an intermediate in glycolysis).Thus, by increasing the efficiency of conversion of sugars to usefulenergy molecules, it is also possible to increase the amount of usefuldegradation products as well.

The isolated nucleic acid sequences of the invention are containedwithin the genome of a Corynebacterium glutamicum strain availablethrough the American Type Culture Collection, given designation ATCC13032. The nucleotide sequence of the isolated C. glutamicum SMP DNAsand the predicted amino acid sequences of the C. glutamicum SMP proteinsare shown in Appendices A and B, respectively. Computational analyseswere performed which classified and/or identified these nucleotidesequences as sequences which encode proteins having a function involvedin the metabolism of carbon compounds such as sugars or in thegeneration of energy molecules by processes such as oxidativephosphorylation in Corynebacterium glutamicum.

The present invention also pertains to proteins which have an amino acidsequence which is substantially homologous to an amino acid sequence ofAppendix B. As used herein, a protein which has an amino acid sequencewhich is substantially homologous to a selected amino acid sequence isleast about 50% homologous to the selected amino acid sequence, e.g.,the entire selected amino acid sequence. A protein which has an aminoacid sequence which is substantially homologous to a selected amino acidsequence can also be least about 50-60%, preferably at least about60-70%, and more preferably at least about 70-80%, 80-90%, or 90-95%,and most preferably at least about 96%, 97%, 98%, 99% or more homologousto the selected amino acid sequence.

An SMP protein or a biologically active portion or fragment thereof ofthe invention can participate in the metabolism of carbon compounds suchas sugars or in the generation of energy molecules (e.g., ATP) byprocesses such as oxidative phosphorylation in Corynebacteriumglutamicum, or can have one or more of the activities set forth in Table1.

Various aspects of the invention are described in further detail in thefollowing subsections:

A. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode SMP polypeptides or biologically active portions thereof, aswell as nucleic acid fragments sufficient for use as hybridizationprobes or primers for the identification or amplification ofSMP-encoding nucleic acid (e.g., SMP DNA). As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (e.g., cDNAor genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. This term also encompassesuntranslated sequence located at both the 3′ and 5′ ends of the codingregion of the gene: at least about 100 nucleotides of sequence upstreamfrom the 5′ end of the coding region and at least about 20 nucleotidesof sequence downstream from the 3′ end of the coding region of the gene.The nucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA. An “isolated” nucleic acid moleculeis one which is separated from other nucleic acid molecules which arepresent in the natural source of the nucleic acid. Preferably, an“isolated” nucleic acid is free of sequences which naturally flank thenucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolated SMPnucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived (e.g, a C. glutamicum cell). Moreover, an“isolated” nucleic acid molecule, such as a DNA molecule, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having a nucleotide sequence of Appendix A, or a portionthereof, can be isolated using standard molecular biology techniques andthe sequence information provided herein. For example, a C. glutamicumSMP DNA can be isolated from a C. glutamicum library using all orportion of one of the sequences of Appendix A as a hybridization probeand standard hybridization techniques (e.g., as described in Sambrook,J., Fritsh, E. F., and Maniatis, T. Molecular Cloning. A LaboratoryManual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989). Moreover, a nucleicacid molecule encompassing all or a portion of one of the sequences ofAppendix A can be isolated by the polymerase chain reaction usingoligonucleotide primers designed based upon this sequence (e.g., anucleic acid molecule encompassing all or a portion of one of thesequences of Appendix A can be isolated by the polymerase chain reactionusing oligonucleotide primers designed based upon this same sequence ofAppendix A). For example, mRNA can be isolated from normal endothelialcells (e.g., by the guanidinium-thiocyanate extraction procedure ofChirgwin et al. (1979) Biochemistry 18: 5294-5299) and DNA can beprepared using reverse transcriptase (e.g., Moloney MLV reversetranscriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reversetranscriptase, available from Seikagaku America, Inc., St. Petersburg,Fla.). Synthetic oligonucleotide primers for polymerase chain reactionamplification can be designed based upon one of the nucleotide sequencesshown in Appendix A. A nucleic acid of the invention can be amplifiedusing cDNA or, alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to an SMP nucleotidesequence can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises one of the nucleotide sequences shown in Appendix A.The sequences of Appendix A correspond to the Corynebacterium glutamicumSMP DNAs of the invention. This DNA comprises sequences encoding SMPproteins (i.e., the “coding region”, indicated in each sequence inAppendix A), as well as 5′ untranslated sequences and 3′ untranslatedsequences, also indicated in Appendix A. Alternatively, the nucleic acidmolecule can comprise only the coding region of any of the sequences inAppendix A.

For the purposes of this application, it will be understood that each ofthe sequences set forth in Appendix A has an identifying RXA, RXN, orRXS number having the designation “RXA,” or “RXS” followed by 5 digits(i.e., RXA00013, RXN00043, or RXS0735). Each of these sequencescomprises up to three parts: a 5′ upstream region, a coding region, anda downstream region. Each of these three regions is identified by thesame RXA, RXN, or RXS designation to eliminate confusion. The recitation“one of the sequences in Appendix A”, then, refers to any of thesequences in Appendix A, which may be distinguished by their differingRXA, RXN, or RXS designations. The coding region of each of thesesequences is translated into a corresponding amino acid sequence, whichis set forth in Appendix B. The sequences of Appendix B are identifiedby the same RXA, RXN, or RXS designations as Appendix A, such that theycan be readily correlated. For example, the amino acid sequence inAppendix B designated RXA00013 is a translation of the coding region ofthe nucleotide sequence of nucleic acid molecule RXA00013 in Appendix A,and the amino acid sequence in Appendix B designated RXN00043 is atranslation of the coding region of the nucleotide sequence of nucleicacid molecule RXN00043 in Appendix A. Each of the RXARXN and RXSnucleotide and amino acid sequences of the invention has also beenassigned a SEQ ID NO, as indicated in Table 1.

Several of the genes of the invention are “F-designated genes”. AnF-designated gene includes those genes set forth in Table 1 which havean ‘F’ in front of the RXA designation. For example, SEQ ID NO:11,designated, as indicated on Table 1, as “F RXA01312”, is an F-designatedgene, as are SEQ ID NOs: 29, 33, and 39 (designated on Table 1 as “FRXA02803”, “F RXA02854”, and “F RXA01365”, respectively).

In one embodiment, the nucleic acid molecules of the present inventionare not intended to include those compiled in Table 2. In the case ofthe dapD gene, a sequence for this gene was published in Wehrmann, A.,et al. (1998) J. Bacteriol. 180(12): 3159-3165. However, the sequenceobtained by the inventors of the present application is significantlylonger than the published version. It is believed that the publishedversion relied on an incorrect start codon, and thus represents only afragment of the actual coding region.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofone of the nucleotide sequences shown in Appendix A, or a portionthereof. A nucleic acid molecule which is complementary to one of thenucleotide sequences shown in Appendix A is one which is sufficientlycomplementary to one of the nucleotide sequences shown in Appendix Asuch that it can hybridize to one of the nucleotide sequences shown inAppendix A, thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the invention comprises a nucleotide sequence which is at least about50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably atleast about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, morepreferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%,92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%,98%, 99% or more homologous to a nucleotide sequence shown in AppendixA, or a portion thereof. Ranges and identity values intermediate to theabove-recited ranges, (e.g., 70-90% identical or 80-95% identical) arealso intended to be encompassed by the present invention. For example,ranges of identity values using a combination of any of the above valuesrecited as upper and/or lower limits are intended to be included. In anadditional preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleotide sequence which hybridizes, e.g.,hybridizes under stringent conditions, to one of the nucleotidesequences shown in Appendix A, or a portion thereof.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the coding region of one of the sequences in Appendix A, forexample a fragment which can be used as a probe or primer or a fragmentencoding a biologically active portion of an SMP protein. The nucleotidesequences determined from the cloning of the SMP genes from C.glutamicum allows for the generation of probes and primers designed foruse in identifying and/or cloning SMP homologues in other cell types andorganisms, as well as SMP homologues from other Corynebacteria orrelated species. The probe/primer typically comprises substantiallypurified oligonucleotide. The oligonucleotide typically comprises aregion of nucleotide sequence that hybridizes under stringent conditionsto at least about 12, preferably about 25, more preferably about 40, 50or 75 consecutive nucleotides of a sense strand of one of the sequencesset forth in Appendix A, an anti-sense sequence of one of the sequencesset forth in Appendix A, or naturally occurring mutants thereof. Primersbased on a nucleotide sequence of Appendix A can be used in PCRreactions to clone SMP homologues. Probes based on the SMP nucleotidesequences can be used to detect transcripts or genomic sequencesencoding the same or homologous proteins. In preferred embodiments, theprobe further comprises a label group attached thereto, e.g. the labelgroup can be a radioisotope, a fluorescent compound, an enzyme, or anenzyme co-factor. Such probes can be used as a part of a diagnostic testkit for identifying cells which misexpress an SMP protein, such as bymeasuring a level of an SMP-encoding nucleic acid in a sample of cells,e.g., detecting SMP mRNA levels or determining whether a genomic SMPgene has been mutated or deleted.

In one embodiment, the nucleic acid molecule of the invention encodes aprotein or portion thereof which includes an amino acid sequence whichis sufficiently homologous to an amino acid sequence of Appendix B suchthat the protein or portion thereof maintains the ability to perform afunction involved in the metabolism of carbon compounds such as sugarsor in the generation of energy molecules (e.g., ATP) by processes suchas oxidative phosphorylation in Corynebacterium glutamicum. As usedherein, the language “sufficiently homologous” refers to proteins orportions thereof which have amino acid sequences which include a minimumnumber of identical or equivalent (e.g., an amino acid residue which hasa similar side chain as an amino acid residue in one of the sequences ofAppendix B) amino acid residues to an amino acid sequence of Appendix Bsuch that the protein or portion thereof is able to perform a functioninvolved in the metabolism of carbon compounds such as sugars or in thegeneration of energy molecules (e.g., ATP) by processes such asoxidative phosphorylation in Corynebacterium glutamicum. Protein membersof such sugar metabolic pathways or energy producing systems, asdescribed herein, may play a role in the production and secretion of oneor more fine chemicals. Examples of such activities are also describedherein. Thus, “the function of an SMP protein” contributes eitherdirectly or indirectly to the yield, production, and/or efficiency ofproduction of one or more fine chemicals. Examples of SMP proteinactivities are set forth in Table 1.

In another embodiment, the protein is at least about 50-60%, preferablyat least about 60-70%, and more preferably at least about 70-80%,80-90%, 90-95%, and most preferably at least about 96%, 97%, 98%, 99% ormore homologous to an entire amino acid sequence of Appendix B.

Portions of proteins encoded by the SMP nucleic acid molecules of theinvention are preferably biologically active portions of one of the SMPproteins. As used herein, the term “biologically active portion of anSMP protein” is intended to include a portion, e.g., a domain/motif, ofan SMP protein that participates in the metabolism of carbon compoundssuch as sugars, or in energy-generating pathways in C. glutamicum, orhas an activity as set forth in Table 1. To determine whether an SMPprotein or a biologically active portion thereof can participate in themetabolism of carbon compounds or in the production of energy-richmolecules in C. glutamicum, an assay of enzymatic activity may beperformed. Such assay methods are well known to those of ordinary skillin the art, as detailed in Example 8 of the Exemplification.

Additional nucleic acid fragments encoding biologically active portionsof an SMP protein can be prepared by isolating a portion of one of thesequences in Appendix B, expressing the encoded portion of the SMPprotein or peptide (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the SMP protein orpeptide.

The invention further encompasses nucleic acid molecules that differfrom one of the nucleotide sequences shown in Appendix A (and portionsthereof) due to degeneracy of the genetic code and thus encode the sameSMP protein as that encoded by the nucleotide sequences shown inAppendix A. In another embodiment, an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having anamino acid sequence shown in Appendix B. In a still further embodiment,the nucleic acid molecule of the invention encodes a full length C.glutamicum protein which is substantially homologous to an amino acidsequence of Appendix B (encoded by an open reading frame shown inAppendix A).

It will be understood by one of ordinary skill in the art that in oneembodiment the sequences of the invention are not meant to include thesequences of the prior art, such as those Genbank sequences set forth inTables 2 or 4 which were available prior to the present invention. Inone embodiment, the invention includes nucleotide and amino acidsequences having a percent identity to a nucleotide or amino acidsequence of the invention which is greater than that of a sequence ofthe prior art (e.g., a Genbank sequence (or the protein encoded by sucha sequence) set forth in Tables 2 or 4). For example, the inventionincludes a nucleotide sequence which is greater than and/or at least 58%identical to the nucleotide sequence designated RXA00014 (SEQ ID NO:41),a nucleotide sequence which is greater than and/or at least % identicalto the nucleotide sequence designated RXA00195 (SEQ ID NO:399), and anucleotide sequence which is greater than and/or at least 42% identicalto the nucleotide sequence designated RXA00196 (SEQ ID NO:401). One ofordinary skill in the art would be able to calculate the lower thresholdof percent identity for any given sequence of the invention by examiningthe GAP-calculated percent identity scores set forth in Table 4 for eachof the three top hits for the given sequence, and by subtracting thehighest GAP-calculated percent identity from 100 percent. One ofordinary skill in the art will also appreciate that nucleic acid andamino acid sequences having percent identities greater than the lowerthreshold so calculated (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even morepreferably at least about 95%, 96%, 97%, 98%, 99% or more identical) arealso encompassed by the invention.

In addition to the C. glutamicum SMP nucleotide sequences shown inAppendix A, it will be appreciated by those of ordinary skill in the artthat DNA sequence polymorphisms that lead to changes in the amino acidsequences of SMP proteins may exist within a population (e.g., the C.glutamicum population). Such genetic polymorphism in the SMP gene mayexist among individuals within a population due to natural variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding an SMP protein,preferably a C. glutamicum SMP protein. Such natural variations cantypically result in 1-5% variance in the nucleotide sequence of the SMPgene. Any and all such nucleotide variations and resulting amino acidpolymorphisms in SMP that are the result of natural variation and thatdo not alter the functional activity of SMP proteins are intended to bewithin the scope of the invention.

Nucleic acid molecules corresponding to natural variants and non-C.glutamicum homologues of the C. glutamicum SMP DNA of the invention canbe isolated based on their homology to the C. glutamicum SMP nucleicacid disclosed herein using the C. glutamicum DNA, or a portion thereof,as a hybridization probe according to standard hybridization techniquesunder stringent hybridization conditions. Accordingly, in anotherembodiment, an isolated nucleic acid molecule of the invention is atleast 15 nucleotides in length and hybridizes under stringent conditionsto the nucleic acid molecule comprising a nucleotide sequence ofAppendix A. In other embodiments, the nucleic acid is at least 30, 50,100, 250 or more nucleotides in length. As used herein, the term“hybridizes under stringent conditions” is intended to describeconditions for hybridization and washing under which nucleotidesequences at least 60% homologous to each other typically remainhybridized to each other. Preferably, the conditions are such thatsequences at least about 65%, more preferably at least about 70%, andeven more preferably at least about 75% or more homologous to each othertypically remain hybridized to each other. Such stringent conditions areknown to those of ordinary skill in the art and can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions toa sequence of Appendix A corresponds to a naturally-occurring nucleicacid molecule. As used herein, a “naturally-occurring” nucleic acidmolecule refers to an RNA or DNA molecule having a nucleotide sequencethat occurs in nature (e.g., encodes a natural protein). In oneembodiment, the nucleic acid encodes a natural C. glutamicum SMPprotein.

In addition to naturally-occurring variants of the SMP sequence that mayexist in the population, one of ordinary skill in the art will furtherappreciate that changes can be introduced by mutation into a nucleotidesequence of Appendix A, thereby leading to changes in the amino acidsequence of the encoded SMP protein, without altering the functionalability of the SMP protein. For example, nucleotide substitutionsleading to amino acid substitutions at “non-essential” amino acidresidues can be made in a sequence of Appendix A. A “non-essential”amino acid residue is a residue that can be altered from the wild-typesequence of one of the SMP proteins (Appendix B) without altering theactivity of said SMP protein, whereas an “essential” amino acid residueis required for SMP protein activity. Other amino acid residues,however, (e.g., those that are not conserved or only semi-conserved inthe domain having SMP activity) may not be essential for activity andthus are likely to be amenable to alteration without altering SMPactivity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding SMP proteins that contain changes in amino acidresidues that are not essential for SMP activity. Such SMP proteinsdiffer in amino acid sequence from a sequence contained in Appendix Byet retain at least one of the SMP activities described herein. In oneembodiment, the isolated nucleic acid molecule comprises a nucleotidesequence encoding a protein, wherein the protein comprises an amino acidsequence at least about 50% homologous to an amino acid sequence ofAppendix B and is capable of participate in the metabolism of carboncompounds such as sugars, or in the biosynthesis of high-energycompounds in C. glutamicum, or has one or more activities set forth inTable 1. Preferably, the protein encoded by the nucleic acid molecule isat least about 50-60% homologous to one of the sequences in Appendix B,more preferably at least about 60-70% homologous to one of the sequencesin Appendix B, even more preferably at least about 70-80%, 80-90%,90-95% homologous to one of the sequences in Appendix B, and mostpreferably at least about 96%, 97%, 98%, or 99% homologous to one of thesequences in Appendix B.

To determine the percent homology of two amino acid sequences (e.g., oneof the sequences of Appendix B and a mutant form thereof) or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of one protein or nucleicacid for optimal alignment with the other protein or nucleic acid). Theamino acid residues or nucleotides at corresponding amino acid positionsor nucleotide positions are then compared. When a position in onesequence (e.g., one of the sequences of Appendix B) is occupied by thesame amino acid residue or nucleotide as the corresponding position inthe other sequence (e.g., a mutant form of the sequence selected fromAppendix B), then the molecules are homologous at that position (i.e.,as used herein amino acid or nucleic acid “homology” is equivalent toamino acid or nucleic acid “identity”). The percent homology between thetwo sequences is a function of the number of identical positions sharedby the sequences (i.e., % homology=# of identical positions/total # ofpositions×100).

An isolated nucleic acid molecule encoding an SMP protein homologous toa protein sequence of Appendix B can be created by introducing one ormore nucleotide substitutions, additions or deletions into a nucleotidesequence of Appendix A such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.Mutations can be introduced into one of the sequences of Appendix A bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in an SMP protein ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of an SMP coding sequence, such asby saturation mutagenesis, and the resultant mutants can be screened foran SMP activity described herein to identify mutants that retain SMPactivity. Following mutagenesis of one of the sequences of Appendix A,the encoded protein can be expressed recombinantly and the activity ofthe protein can be determined using, for example, assays describedherein (see Example 8 of the Exemplification).

In addition to the nucleic acid molecules encoding SMP proteinsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence which is complementary to a“sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded DNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire SMP coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding an SMPprotein. The term “coding region” refers to the region of the nucleotidesequence comprising codons which are translated into amino acid residues(e.g., the entire coding region of NO. 3 (RXA01626) comprisesnucleotides 1 to 345). In another embodiment, the antisense nucleic acidmolecule is antisense to a “noncoding region” of the coding strand of anucleotide sequence encoding SMP. The term “noncoding region” refers to5′ and 3′ sequences which flank the coding region that are nottranslated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

Given the coding strand sequences encoding SMP disclosed herein (e.g.,the sequences set forth in Appendix A), antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule can be complementaryto the entire coding region of SMP mRNA, but more preferably is anoligonucleotide which is antisense to only a portion of the coding ornoncoding region of SMP mRNA. For example, the antisense oligonucleotidecan be complementary to the region surrounding the translation startsite of SMP mRNA. An antisense oligonucleotide can be, for example,about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. Anantisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the antisense nucleic acid include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine.Alternatively, the antisense nucleic acid can be produced biologicallyusing an expression vector into which a nucleic acid has been subclonedin an antisense orientation (i.e., RNA transcribed from the insertednucleic acid will be of an antisense orientation to a target nucleicacid of interest, described further in the following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a cell or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding an SMP proteinto thereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic promoter arepreferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleaveSMP mRNA transcripts to thereby inhibit translation of SMP mRNA. Aribozyme having specificity for an SMP-encoding nucleic acid can bedesigned based upon the nucleotide sequence of an SMP cDNA disclosedherein (i.e., SEQ ID NO. 3 (RXA01626) in Appendix A). For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in an SMP-encoding mRNA. See, e.g.,Cech et al. U.S. Pat. No. 4,987,071 and Cech et al. U.S. Pat. No.5,116,742. Alternatively, SMP mRNA can be used to select a catalytic RNAhaving a specific ribonuclease activity from a pool of RNA molecules.See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

Alternatively, SMP gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of an SMPnucleotide sequence (e.g., an SMP promoter and/or enhancers) to formtriple helical structures that prevent transcription of an SMP gene intarget cells. See generally, Helene, C. (1991) Anticancer Drug Des.6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36;and Maher, L. J. (1992) Bioassays 14(12):807-15.

B. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding an SMP protein(or a portion thereof). As used herein, the term “vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments can be ligated. Another type of vector is a viral vector,wherein additional DNA segments can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells.Preferred regulatory sequences are, for example, promoters such as cos-,tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacI^(q)-, T7-, T5-,T3-, gal-, trc-, ara-, SP6-, arny, SPO2, λ-P_(R)- or λP_(L), which areused preferably in bacteria. Additional regulatory sequences are, forexample, promoters from yeasts and fungi, such as ADC1, MFα, AC, P-60,CYC1, GAPDH, TEF, rp28, ADH, promoters from plants such as CaMV/35S,SSU, OCS, lib4, usp, STLS1, B33, nos or ubiquitin- orphaseolin-promoters. It is also possible to use artificial promoters. Itwill be appreciated by those of ordinary skill in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, etc. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein (e.g., SMP proteins, mutant forms of SMP proteins, fusionproteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of SMP proteins in prokaryotic or eukaryotic cells. Forexample, SMP genes can be expressed in bacterial cells such as C.glutamicum, insect cells (using baculovirus expression vectors), yeastand other fungal cells (see Romanos, M. A. et al. (1992) “Foreign geneexpression in yeast: a review”, Yeast 8: 423-488; van den Hondel, C. A.M. J. J. et al. (1991) “Heterologous gene expression in filamentousfungi” in: More Gene Manipulations in Fungi, J. W. Bennet & L. L.Lasure, eds., p. 396-428: Academic Press: San Diego; and van den Hondel,C. A. M. J. J. & Punt, P. J. (1991) “Gene transfer systems and vectordevelopment for filamentous fungi, in: Applied Molecular Genetics ofFungi, Peberdy, J. F. et al., eds., p. 1-28, Cambridge University Press:Cambridge), algae and multicellular plant cells (see Schmidt, R. andWillmitzer, L. (1988) High efficiency Agrobacterium tumefaciens-mediatedtransformation of Arabidopsis thaliana leaf and cotyledon explants”Plant Cell Rep: 583-586), or mammalian cells. Suitable host cells arediscussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion proteins. Fusion vectors add anumber of amino acids to a protein encoded therein, usually to the aminoterminus of the recombinant protein but also to the C-terminus or fusedwithin suitable regions in the proteins. Such fusion vectors typicallyserve three purposes: 1) to increase expression of recombinant protein;2) to increase the solubility of the recombinant protein; and 3) to aidin the purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. In one embodiment,the coding sequence of the SMP protein is cloned into a pGEX expressionvector to create a vector encoding a fusion protein comprising, from theN-terminus to the C-terminus, GST-thrombin cleavage site-X protein. Thefusion protein can be purified by affinity chromatography usingglutathione-agarose resin. Recombinant SMP protein unfused to GST can berecovered by cleavage of the fusion protein with thrombin.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315), pLG338, pACYC184,pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200,pUR290, pIN-III113-B1, λgt11, pBdC1, and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 60-89; and Pouwels et al., eds. (1985) CloningVectors. Elsevier: New York IBSN 0 444 904018). Target gene expressionfrom the pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident λ prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter. For transformation of other varietiesof bacteria, appropriate vectors may be selected. For example, theplasmids pIJ101, pIJ364, pIJ702 and pIJ361 are known to be useful intransforming Streptomyces, while plasmids pUB110, pC194, or pBD214 aresuited for transformation of Bacillus species. Several plasmids of usein the transfer of genetic information into Corynebacterium includepHM1519, pBL1, pSA77, or pAJ667 (Pouwels et al., eds. (1985) CloningVectors. Elsevier: New York IBSN 0 444 904018).

One strategy to maximize recombinant protein expression is to expressthe protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in the bacterium chosen for expression, such asC. glutamicum (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences of the invention can be carried outby standard DNA synthesis techniques.

In another embodiment, the SMP protein expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerevisiae include pYepSec1 (Baldari, et al., (1987) Embo J. 6:229-234),2μ, pAG-1, Yep6, Yep13, pEMBLYe23, pMFa (Kurjan and Herskowitz, (1982)Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), andpYES2 (Invitrogen Corporation, San Diego, Calif.). Vectors and methodsfor the construction of vectors appropriate for use in other fungi, suchas the filamentous fungi, include those detailed in: van den Hondel, C.A. M. J. J. & Punt, P. J. (1991) “Gene transfer systems and vectordevelopment for filamentous fungi, in: Applied Molecular Genetics ofFungi, J. F. Peberdy, et al., eds., p. 1-28, Cambridge University Press:Cambridge, and Pouwels et al., eds. (1985) Cloning Vectors. Elsevier:New York (IBSN 0 444 904018).

Alternatively, the SMP proteins of the invention can be expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

In another embodiment, the SMP proteins of the invention may beexpressed in unicellular plant cells (such as algae) or in plant cellsfrom higher plants (e.g., the spermatophytes, such as crop plants).Examples of plant expression vectors include those detailed in: Becker,D., Kemper, E., Schell, J. and Masterson, R. (1992) “New plant binaryvectors with selectable markers located proximal to the left border”,Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W. (1984) “BinaryAgrobacterium vectors for plant transformation”, Nucl. Acid. Res. 12:8711-8721, and include pLGV23, pGHlac+, pBIN19, pAK2004, and pDH51(Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0444 904018).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to SMP mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosenwhich direct the continuous expression of the antisense RNA molecule ina variety of cell types, for instance viral promoters and/or enhancers,or regulatory sequences can be chosen which direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub, H. et al., Antisense RNAas a molecular tool for genetic analysis, Reviews—Trends in Genetics,Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, anSMP protein can be expressed in bacterial cells such as C. glutamicum,insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells). Other suitable host cells are known to one ofordinary skill in the art. Microorganisms related to Corynebacteriumglutamicum which may be conveniently used as host cells for the nucleicacid and protein molecules of the invention are set forth in Table 3.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection”, “conjugation” and“transduction” are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., linear DNA or RNA(e.g., a linearized vector or a gene construct alone without a vector)or nucleic acid in the form of a vector (e.g., a plasmid, phage,phasmid, phagemid, transposon or other DNA) into a host cell, includingcalcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,chemical-mediated transfer, or electroporation. Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et al.(Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding an SMP protein or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by, for example, drug selection (e.g., cells thathave incorporated the selectable marker gene will survive, while theother cells die).

To create a homologous recombinant microorganism, a vector is preparedwhich contains at least a portion of an SMP gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the SMP gene. Preferably, this SMP gene is aCorynebacterium glutamicum SMP gene, but it can be a homologue from arelated bacterium or even from a mammalian, yeast, or insect source. Ina preferred embodiment, the vector is designed such that, uponhomologous recombination, the endogenous SMP gene is functionallydisrupted (i.e., no longer encodes a functional protein; also referredto as a “knock out” vector). Alternatively, the vector can be designedsuch that, upon homologous recombination, the endogenous SMP gene ismutated or otherwise altered but still encodes functional protein (e.g.,the upstream regulatory region can be altered to thereby alter theexpression of the endogenous SMP protein). In the homologousrecombination vector, the altered portion of the SMP gene is flanked atits 5′ and 3′ ends by additional nucleic acid of the SMP gene to allowfor homologous recombination to occur between the exogenous SMP genecarried by the vector and an endogenous SMP gene in a microorganism. Theadditional flanking SMP nucleic acid is of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several kilobases of flanking DNA (both at the 5′ and 3′ ends) areincluded in the vector (see e.g., Thomas, K. R., and Capecchi, M. R.(1987) Cell 51: 503 for a description of homologous recombinationvectors). The vector is introduced into a microorganism (e.g., byelectroporation) and cells in which the introduced SMP gene hashomologously recombined with the endogenous SMP gene are selected, usingart-known techniques.

In another embodiment, recombinant microorganisms can be produced whichcontain selected systems which allow for regulated expression of theintroduced gene. For example, inclusion of an SMP gene on a vectorplacing it under control of the lac operon permits expression of the SMPgene only in the presence of IPTG. Such regulatory systems are wellknown in the art.

In another embodiment, an endogenous SMP gene in a host cell isdisrupted (e.g., by homologous recombination or other genetic meansknown in the art) such that expression of its protein product does notoccur. In another embodiment, an endogenous or introduced SMP gene in ahost cell has been altered by one or more point mutations, deletions, orinversions, but still encodes a functional SMP protein. In still anotherembodiment, one or more of the regulatory regions (e.g., a promoter,repressor, or inducer) of an SMP gene in a microorganism has beenaltered (e.g., by deletion, truncation, inversion, or point mutation)such that the expression of the SMP gene is modulated. One of ordinaryskill in the art will appreciate that host cells containing more thanone of the described SMP gene and protein modifications may be readilyproduced using the methods of the invention, and are meant to beincluded in the present invention.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) an SMP protein.Accordingly, the invention further provides methods for producing SMPproteins using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding an SMP protein has beenintroduced, or into which genome has been introduced a gene encoding awild-type or altered SMP protein) in a suitable medium until SMP proteinis produced. In another embodiment, the method further comprisesisolating SMP proteins from the medium or the host cell.

C. Isolated SMP Proteins

Another aspect of the invention pertains to isolated SMP proteins, andbiologically active portions thereof. An “isolated” or “purified”protein or biologically active portion thereof is substantially free ofcellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof SMP protein in which the protein is separated from cellularcomponents of the cells in which it is naturally or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of SMP protein having less thanabout 30% (by dry weight) of non-SMP protein (also referred to herein asa “contaminating protein”), more preferably less than about 20% ofnon-SMP protein, still more preferably less than about 10% of non-SMPprotein, and most preferably less than about 5% non-SMP protein. Whenthe SMP protein or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the protein preparation. The language “substantially free ofchemical precursors or other chemicals” includes preparations of SMPprotein in which the protein is separated from chemical precursors orother chemicals which are involved in the synthesis of the protein. Inone embodiment, the language “substantially free of chemical precursorsor other chemicals” includes preparations of SMP protein having lessthan about 30% (by dry weight) of chemical precursors or non-SMPchemicals, more preferably less than about 20% chemical precursors ornon-SMP chemicals, still more preferably less than about 10% chemicalprecursors or non-SMP chemicals, and most preferably less than about 5%chemical precursors or non-SMP chemicals. In preferred embodiments,isolated proteins or biologically active portions thereof lackcontaminating proteins from the same organism from which the SMP proteinis derived. Typically, such proteins are produced by recombinantexpression of, for example, a C. glutamicum SMP protein in amicroorganism such as C. glutamicum.

An isolated SMP protein or a portion thereof of the invention canparticipate in the metabolism of carbon compounds such as sugars, or inthe production of energy compounds (e.g., by oxidative phosphorylation)utilized to drive unfavorable metabolic pathways, or has one or more ofthe activities set forth in Table 1. In preferred embodiments, theprotein or portion thereof comprises an amino acid sequence which issufficiently homologous to an amino acid sequence of Appendix B suchthat the protein or portion thereof maintains the ability to perform afunction involved in the metabolism of carbon compounds such as sugarsor in the generation of energy molecules by processes such as oxidativephosphorylation in Corynebacterium glutamicum. The portion of theprotein is preferably a biologically active portion as described herein.In another preferred embodiment, an SMP protein of the invention has anamino acid sequence shown in Appendix B. In yet another preferredembodiment, the SMP protein has an amino acid sequence which is encodedby a nucleotide sequence which hybridizes, e.g., hybridizes understringent conditions, to a nucleotide sequence of Appendix A. In stillanother preferred embodiment, the SMP protein has an amino acid sequencewhich is encoded by a nucleotide sequence that is at least about 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at leastabout 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, morepreferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%,92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%,98%, 99% or more homologous to one of the nucleic acid sequences ofAppendix A, or a portion thereof. Ranges and identity valuesintermediate to the above-recited values, (e.g., 70-90% identical or80-95% identical) are also intended to be encompassed by the presentinvention. For example, ranges of identity values using a combination ofany of the above values recited as upper and/or lower limits areintended to be included. The preferred SMP proteins of the presentinvention also preferably possess at least one of the SMP activitiesdescribed herein. For example, a preferred SMP protein of the presentinvention includes an amino acid sequence encoded by a nucleotidesequence which hybridizes, e.g., hybridizes under stringent conditions,to a nucleotide sequence of Appendix A, and which can perform a functioninvolved in the metabolism of carbon compounds such as sugars or in thegeneration of energy molecules (e.g., ATP) by processes such asoxidative phosphorylation in Corynebacterium glutamicum, or which hasone or more of the activities set forth in Table 1.

In other embodiments, the SMP protein is substantially homologous to anamino acid sequence of Appendix B and retains the functional activity ofthe protein of one of the sequences of Appendix B yet differs in aminoacid sequence due to natural variation or mutagenesis, as described indetail in subsection I above. Accordingly, in another embodiment, theSMP protein is a protein which comprises an amino acid sequence which isat least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%,preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%,96%, 97%, 98%, 99% or more homologous to an entire amino acid sequenceof Appendix B and which has at least one of the SMP activities describedherein. Ranges and identity values intermediate to the above-recitedvalues, (e.g., 70-90% identical or 80-95% identical) are also intendedto be encompassed by the present invention. For example, ranges ofidentity values using a combination of any of the above values recitedas upper and/or lower limits are intended to be included. In anotherembodiment, the invention pertains to a full length C. glutamicumprotein which is substantially homologous to an entire amino acidsequence of Appendix B.

Biologically active portions of an SMP protein include peptidescomprising amino acid sequences derived from the amino acid sequence ofan SMP protein, e.g., the an amino acid sequence shown in Appendix B orthe amino acid sequence of a protein homologous to an SMP protein, whichinclude fewer amino acids than a full length SMP protein or the fulllength protein which is homologous to an SMP protein, and exhibit atleast one activity of an SMP protein. Typically, biologically activeportions (peptides, e.g., peptides which are, for example, 5, 10, 15,20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length)comprise a domain or motif with at least one activity of an SMP protein.Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the activities described herein.Preferably, the biologically active portions of an SMP protein includeone or more selected domains/motifs or portions thereof havingbiological activity.

SMP proteins are preferably produced by recombinant DNA techniques. Forexample, a nucleic acid molecule encoding the protein is cloned into anexpression vector (as described above), the expression vector isintroduced into a host cell (as described above) and the SMP protein isexpressed in the host cell. The SMP protein can then be isolated fromthe cells by an appropriate purification scheme using standard proteinpurification techniques. Alternative to recombinant expression, an SMPprotein, polypeptide, or peptide can be synthesized chemically usingstandard peptide synthesis techniques. Moreover, native SMP protein canbe isolated from cells (e.g., endothelial cells), for example using ananti-SMP antibody, which can be produced by standard techniquesutilizing an SMP protein or fragment thereof of this invention.

The invention also provides SMP chimeric or fusion proteins. As usedherein, an SMP “chimeric protein” or “fusion protein” comprises an SMPpolypeptide operatively linked to a non-SMP polypeptide. An “SMPpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to an SMP protein, whereas a “non-SMP polypeptide” refersto a polypeptide having an amino acid sequence corresponding to aprotein which is not substantially homologous to the SMP protein, e.g.,a protein which is different from the SMP protein and which is derivedfrom the same or a different organism. Within the fusion protein, theterm “operatively linked” is intended to indicate that the SMPpolypeptide and the non-SMP polypeptide are fused in-frame to eachother. The non-SMP polypeptide can be fused to the N-terminus orC-terminus of the SMP polypeptide. For example, in one embodiment thefusion protein is a GST SMP fusion protein in which the SMP sequencesare fused to the C-terminus of the GST sequences. Such fusion proteinscan facilitate the purification of recombinant SMP proteins. In anotherembodiment, the fusion protein is an SMP protein containing aheterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian host cells), expression and/or secretion of an SMPprotein can be increased through use of a heterologous signal sequence.

Preferably, an SMP chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, Ausubel et al., eds. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). AnSMP-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the SMP protein.

Homologues of the SMP protein can be generated by mutagenesis, e.g.,discrete point mutation or truncation of the SMP protein. As usedherein, the term “homologue” refers to a variant form of the SMP proteinwhich acts as an agonist or antagonist of the activity of the SMPprotein. An agonist of the SMP protein can retain substantially thesame, or a subset, of the biological activities of the SMP protein. Anantagonist of the SMP protein can inhibit one or more of the activitiesof the naturally occurring form of the SMP protein, by, for example,competitively binding to a downstream or upstream member of the sugarmolecule metabolic cascade or the energy-producing pathway whichincludes the SMP protein.

In an alternative embodiment, homologues of the SMP protein can beidentified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of the SMP protein for SMP protein agonist orantagonist activity. In one embodiment, a variegated library of SMPvariants is generated by combinatorial mutagenesis at the nucleic acidlevel and is encoded by a variegated gene library. A variegated libraryof SMP variants can be produced by, for example, enzymatically ligatinga mixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential SMP sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of SMP sequences therein.There are a variety of methods which can be used to produce libraries ofpotential SMP homologues from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be performed in anautomatic DNA synthesizer, and the synthetic gene then ligated into anappropriate expression vector. Use of a degenerate set of genes allowsfor the provision, in one mixture, of all of the sequences encoding thedesired set of potential SMP sequences. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acid Res. 11:477.

In addition, libraries of fragments of the SMP protein coding can beused to generate a variegated population of SMP fragments for screeningand subsequent selection of homologues of an SMP protein. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of an SMP coding sequence with anuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the SMP protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of SMP homologues. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify SMP homologues (Arkin and Yourvan (1992) PNAS 89:7811-7815;Delgrave et al. (1993) Protein Engineering 6(3):327-331).

In another embodiment, cell based assays can be exploited to analyze avariegated SMP library, using methods well known in the art.

D. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, fusionproteins, primers, vectors, and host cells described herein can be usedin one or more of the following methods: identification of C. glutamicumand related organisms; mapping of genomes of organisms related to C.glutamicum; identification and localization of C. glutamicum sequencesof interest; evolutionary studies; determination of SMP protein regionsrequired for function; modulation of an SMP protein activity; modulationof the metabolism of one or more sugars; modulation of high-energymolecule production in a cell (i.e., ATP, NADPH); and modulation ofcellular production of a desired compound, such as a fine chemical.

The SMP nucleic acid molecules of the invention have a variety of uses.First, they may be used to identify an organism as being Corynebacteriumglutamicum or a close relative thereof. Also, they may be used toidentify the presence of C. glutamicum or a relative thereof in a mixedpopulation of microorganisms. The invention provides the nucleic acidsequences of a number of C. glutamicum genes; by probing the extractedgenomic DNA of a culture of a unique or mixed population ofmicroorganisms under stringent conditions with a probe spanning a regionof a C. glutamicum gene which is unique to this organism, one canascertain whether this organism is present. Although Corynebacteriumglutamicum itself is nonpathogenic, it is related to pathogenic species,such as Corynebacterium diphtheriae. Corynebacterium diphtheriae is thecausative agent of diphtheria, a rapidly developing, acute, febrileinfection which involves both local and systemic pathology. In thisdisease, a local lesion develops in the upper respiratory tract andinvolves necrotic injury to epithelial cells; the bacilli secrete toxinwhich is disseminated through this lesion to distal susceptible tissuesof the body. Degenerative changes brought about by the inhibition ofprotein synthesis in these tissues, which include heart, muscle,peripheral nerves, adrenals, kidneys, liver and spleen, result in thesystemic pathology of the disease. Diphtheria continues to have highincidence in many parts of the world, including Africa, Asia, EasternEurope and the independent states of the former Soviet Union. An ongoingepidemic of diphtheria in the latter two regions has resulted in atleast 5,000 deaths since 1990.

In one embodiment, the invention provides a method of identifying thepresence or activity of Cornyebacterium diphtheriae in a subject. Thismethod includes detection of one or more of the nucleic acid or aminoacid sequences of the invention (e.g., the sequences set forth inAppendix A or Appendix B) in a subject, thereby detecting the presenceor activity of Corynebacterium diphtheriae in the subject. C. glutamicumand C. diphtheriae are related bacteria, and many of the nucleic acidand protein molecules in C. glutamicum are homologous to C. diphtheriaenucleic acid and protein molecules, and can therefore be used to detectC. diphtheriae in a subject.

The nucleic acid and protein molecules of the invention may also serveas markers for specific regions of the genome. This has utility not onlyin the mapping of the genome, but also for functional studies of C.glutamicum proteins. For example, to identify the region of the genometo which a particular C. glutamicum DNA-binding protein binds, the C.glutamicum genome could be digested, and the fragments incubated withthe DNA-binding protein. Those which bind the protein may beadditionally probed with the nucleic acid molecules of the invention,preferably with readily detectable labels; binding of such a nucleicacid molecule to the genome fragment enables the localization of thefragment to the genome map of C. glutamicum, and, when performedmultiple times with different enzymes, facilitates a rapid determinationof the nucleic acid sequence to which the protein binds. Further, thenucleic acid molecules of the invention may be sufficiently homologousto the sequences of related species such that these nucleic acidmolecules may serve as markers for the construction of a genomic map inrelated bacteria, such as Brevibacterium lactofermentum.

The SMP nucleic acid molecules of the invention are also useful forevolutionary and protein structural studies. The metabolic andenergy-releasing processes in which the molecules of the inventionparticipate are utilized by a wide variety of prokaryotic and eukaryoticcells; by comparing the sequences of the nucleic acid molecules of thepresent invention to those encoding similar enzymes from otherorganisms, the evolutionary relatedness of the organisms can beassessed. Similarly, such a comparison permits an assessment of whichregions of the sequence are conserved and which are not, which may aidin determining those regions of the protein which are essential for thefunctioning of the enzyme. This type of determination is of value forprotein engineering studies and may give an indication of what theprotein can tolerate in terms of mutagenesis without losing function.

Manipulation of the SMP nucleic acid molecules of the invention mayresult in the production of SMP proteins having functional differencesfrom the wild-type SMP proteins. These proteins may be improved inefficiency or activity, may be present in greater numbers in the cellthan is usual, or may be decreased in efficiency or activity.

The invention provides methods for screening molecules which modulatethe activity of an SMP protein, either by interacting with the proteinitself or a substrate or binding partner of the SMP protein, or bymodulating the transcription or translation of an SMP nucleic acidmolecule of the invention. In such methods, a microorganism expressingone or more SMP proteins of the invention is contacted with one or moretest compounds, and the effect of each test compound on the activity orlevel of expression of the SMP protein is assessed.

There are a number of mechanisms by which the alteration of an SMPprotein of the invention may directly affect the yield, production,and/or efficiency of production of a fine chemical from a C. glutamicumstrain incorporating such an altered protein. The degradation ofhigh-energy carbon molecules such as sugars, and the conversion ofcompounds such as NADH and FADH₂ to more useful forms via oxidativephosphorylation results in a number of compounds which themselves may bedesirable fine chemicals, such as pyruvate, ATP, NADH, and a number ofintermediate sugar compounds. Further, the energy molecules (such asATP) and the reducing equivalents (such as NADH or NADPH) produced bythese metabolic pathways are utilized in the cell to drive reactionswhich would otherwise be energetically unfavorable. Such unfavorablereactions include many biosynthetic pathways for fine chemicals. Byimproving the ability of the cell to utilize a particular sugar (e.g.,by manipulating the genes encoding enzymes involved in the degradationand conversion of that sugar into energy for the cell), one may increasethe amount of energy available to permit unfavorable, yet desiredmetabolic reactions (e.g., the biosynthesis of a desired fine chemical)to occur.

Further, modulation of one or more pathways involved in sugarutilization permits optimization of the conversion of the energycontained within the sugar molecule to the production of one or moredesired fine chemicals. For example, by reducing the activity of enzymesinvolved in, for example, gluconeogenesis, more ATP is available todrive desired biochemical reactions (such as fine chemical biosyntheses)in the cell. Also, the overall production of energy molecules fromsugars may be modulated to ensure that the cell maximizes its energyproduction from each sugar molecule. Inefficient sugar utilization canlead to excess CO₂ production and excess energy, which may result infutile metabolic cycles. By improving the metabolism of sugar molecules,the cell should be able to function more efficiently, with a need forfewer carbon molecules. This should result in an improved fine chemicalproduct: sugar molecule ratio (improved carbon yield), and permits adecrease in the amount of sugars that must be added to the medium inlarge-scale fermentor culture of such engineered C. glutamicum.

The mutagenesis of one or more SMP genes of the invention may alsoresult in SMP proteins having altered activities which indirectly impactthe production of one or more desired fine chemicals from C. glutamicum.For example, by increasing the efficiency of utilization of one or moresugars (such that the conversion of the sugar to useful energy moleculesis improved), or by increasing the efficiency of conversion of reducingequivalents to useful energy molecules (e.g., by improving theefficiency of oxidative phosphorylation, or the activity of the ATPsynthase), one can increase the amount of these high-energy compoundsavailable to the cell to drive normally unfavorable metabolic processes.These processes include the construction of cell walls, transcription,translation, and the biosynthesis of compounds necessary for growth anddivision of the cells (e.g., nucleotides, amino acids, vitamins, lipids,etc.) (Lengeler et al. (1999) Biology of Prokaryotes, Thieme Verlag:Stuttgart, p. 88-109; 913-918; 875-899). By improving the growth andmultiplication of these engineered cells, it is possible to increaseboth the viability of the cells in large-scale culture, and also toimprove their rate of division, such that a relatively larger number ofcells can survive in fermentor culture. The yield, production, orefficiency of production may be increased, at least due to the presenceof a greater number of viable cells, each producing the desired finechemical.

Further, many of the degradation products produced during sugarmetabolism are themselves utilized by the cell as precursors orintermediates for the production of a number of other useful compounds,some of which are fine chemicals. For example, pyruvate is convertedinto the amino acid alanine, and ribose-5-phosphate is an integral partof, for example, nucleotide molecules. The amount and efficiency ofsugar metabolism, then, has a profound effect on the availability ofthese degradation products in the cell. By increasing the ability of thecell to process sugars, either in terms of efficiency of existingpathways (e.g., by engineering enzymes involved in these pathways suchthat they are optimized in activity), or by increasing the availabilityof the enzymes involved in such pathways (e.g., by increasing the numberof these enzymes present in the cell), it is possible to also increasethe availability of these degradation products in the cell, which shouldin turn increase the production of many different other desirablecompounds in the cell (e.g., fine chemicals).

The aforementioned mutagenesis strategies for SMP proteins to result inincreased yields of a fine chemical from C. glutamicum are not meant tobe limiting; variations on these strategies will be readily apparent toone of ordinary skill in the art. Using such strategies, andincorporating the mechanisms disclosed herein, the nucleic acid andprotein molecules of the invention may be utilized to generate C.glutamicum or related strains of bacteria expressing mutated SMP nucleicacid and protein molecules such that the yield, production, and/orefficiency of production of a desired compound is improved. This desiredcompound may be any product produced by C. glutamicum, which includesthe final products of biosynthesis pathways and intermediates ofnaturally-occurring metabolic pathways, as well as molecules which donot naturally occur in the metabolism of C. glutamicum, but which areproduced by a C. glutamicum strain of the invention.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patent applications, patents, published patent applications, Tables,Appendices, and the sequence listing cited throughout this applicationare hereby incorporated by reference.

EXEMPLIFICATION Example 1 Preparation of Total Genomic DNA ofCorynebacterium glutamicum ATCC 13032

A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnightat 30° C. with vigorous shaking in BHI medium (Difco). The cells wereharvested by centrifugation, the supernatant was discarded and the cellswere resuspended in 5 ml buffer-I (5% of the original volume of theculture—all indicated volumes have been calculated for 100 ml of culturevolume). Composition of buffer-I: 140.34 g/l sucrose, 2.46 g/lMgSO₄×7H₂O, 10 ml/l KH₂PO₄ solution (100 g/l, adjusted to pH 6.7 withKOH), 50 ml/l M12 concentrate (10 g/l (NH₄)₂SO₄, 1 g/l NaCl, 2 g/lMgSO₄×7H ₂O, 0.2 g/l CaCl₂, 0.5 g/l yeast extract (Difco), 10 ml/ltrace-elements-mix (200 mg/l FeSO₄×H₂O, 10 mg/l ZnSO₄×7 H₂O, 3 mg/lMnCl₂×4 H₂O, 30 mg/l H₃BO₃ 20 mg/l CoCl₂×6H₂O, 1 mg/l NiCl₂×6H₂O, 3 mg/lNa₂MoO₄×2H₂O, 500 mg/l complexing agent (EDTA or critic acid), 100 ml/lvitamins-mix (0.2 mg/l biotin, 0.2 mg/l folic acid, 20 mg/l p-aminobenzoic acid, 20 mg/l riboflavin, 40 mg/l ca-panthothenate, 140 mg/lnicotinic acid, 40 mg/l pyridoxole hydrochloride, 200 mg/lmyo-inositol). Lysozyme was added to the suspension to a finalconcentration of 2.5 mg/ml. After an approximately 4 h incubation at 37°C., the cell wall was degraded and the resulting protoplasts areharvested by centrifugation. The pellet was washed once with 5 mlbuffer-I and once with 5 ml TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8).The pellet was resuspended in 4 ml TE-buffer and 0.5 ml SDS solution(10%) and 0.5 ml NaCl solution (5 M) are added. After adding ofproteinase K to a final concentration of 200 μg/ml, the suspension isincubated for ca. 18 h at 37° C. The DNA was purified by extraction withphenol, phenol-chloroform-isoamylalcohol and chloroform-isoamylalcoholusing standard procedures. Then, the DNA was precipitated by adding 1/50volume of 3 M sodium acetate and 2 volumes of ethanol, followed by a 30min incubation at −20° C. and a 30 min centrifugation at 12,000 rpm in ahigh speed centrifuge using a SS34 rotor (Sorvall). The DNA wasdissolved in 1 ml TE-buffer containing 20 μg/ml RNaseA and dialysed at4° C. against 1000 ml TE-buffer for at least 3 hours. During this time,the buffer was exchanged 3 times. To aliquots of 0.4 ml of the dialysedDNA solution, 0.4 ml of 2 M LiCl and 0.8 ml of ethanol are added. Aftera 30 min incubation at −20° C., the DNA was collected by centrifugation(13,000 rpm, Biofuge Fresco, Heraeus, Hanau, Germany). The DNA pelletwas dissolved in TE-buffer. DNA prepared by this procedure could be usedfor all purposes, including southern blotting or construction of genomiclibraries.

Example 2 Construction of Genomic Libraries in Escherichia coli ofCorynebacterium glutamicum ATCC13032

Using DNA prepared as described in Example 1, cosmid and plasmidlibraries were constructed according to known and well establishedmethods (see e.g., Sambrook, J. et al. (1989) “Molecular Cloning: ALaboratory Manual”, Cold Spring Harbor Laboratory Press, or Ausubel, F.M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley &Sons.)

Any plasmid or cosmid could be used. Of particular use were the plasmidspBR322 (Sutcliffe, J. G. (1979) Proc. Natl. Acad. Sci. USA,75:3737-3741); pACYC177 (Change & Cohen (1978) J. Bacteriol134:1141-1156), plasmids of the pBS series (pBSSK+, pBSSK− and others;Stratagene, LaJolla, USA), or cosmids as SuperCos1 (Stratagene, LaJolla,USA) or Lorist6 (Gibson, T. J., Rosenthal A. and Waterson, R. H. (1987)Gene 53:283-286. Gene libraries specifically for use in C. glutamicummay be constructed using plasmid pSL109 (Lee, H.-S. and A. J. Sinskey(1994) J. Microbiol. Biotechnol. 4: 256-263).

Example 3 DNA Sequencing and Computational Functional Analysis

Genomic libraries as described in Example 2 were used for DNA sequencingaccording to standard methods, in particular by the chain terminationmethod using ABI377 sequencing machines (see e.g., Fleischman, R. D. etal. (1995) “Whole-genome Random Sequencing and Assembly of HaemophilusInfluenzae Rd., Science, 269:496-512). Sequencing primers with thefollowing nucleotide sequences were used: 5′-GGAAACAGTATGACCATG-3′ or5′-GTAAAACGACGGCCAGT-3′.

Example 4 In Vivo Mutagenesis

In vivo mutagenesis of Corynebacterium glutamicum can be performed bypassage of plasmid (or other vector) DNA through E. coli or othermicroorganisms (e.g. Bacillus spp. or yeasts such as Saccharomycescerevisiae) which are impaired in their capabilities to maintain theintegrity of their genetic information. Typical mutator strains havemutations in the genes for the DNA repair system (e.g., mutHLS, mutD,mutT, etc.; for reference, see Rupp, W. D. (1996) DNA repair mechanisms,in: Escherichia coli and Salmonella, p. 2277-2294, ASM: Washington.)Such strains are well known to those of ordinary skill in the art. Theuse of such strains is illustrated, for example, in Greener, A. andCallahan, M. (1994) Strategies 7: 32-34.

Example 5 DNA Transfer Between Escherichia coli and Corynebacteriumglutamicum

Several Corynebacterium and Brevibacterium species contain endogenousplasmids (as e.g., pHM1519 or pBL1) which replicate autonomously (forreview see, e.g., Martin, J. F. et al. (1987) Biotechnology, 5:137-146).Shuttle vectors for Escherichia coli and Corynebacterium glutamicum canbe readily constructed by using standard vectors for E. coli (Sambrook,J. et al. (1989), “Molecular Cloning: A Laboratory Manual”, Cold SpringHarbor Laboratory Press or Ausubel, F. M. et al. (1994) “CurrentProtocols in Molecular Biology”, John Wiley & Sons) to which a origin orreplication for and a suitable marker from Corynebacterium glutamicum isadded. Such origins of replication are preferably taken from endogenousplasmids isolated from Corynebacterium and Brevibacterium species. Ofparticular use as transformation markers for these species are genes forkanamycin resistance (such as those derived from the Tn5 or Tn903transposons) or chloramphenicol (Winnacker, E. L. (1987) “From Genes toClones—Introduction to Gene Technology, VCH, Weinheim). There arenumerous examples in the literature of the construction of a widevariety of shuttle vectors which replicate in both E. coli and C.glutamicum, and which can be used for several purposes, including geneoverexpression (for reference, see e.g., Yoshihama, M. et al. (1985) J.Bacteriol. 162:591-597, Martin J. F. et al. (1987) Biotechnology,5:137-146 and Eikmanns, B. J. et al. (1991) Gene, 102:93-98).

Using standard methods, it is possible to clone a gene of interest intoone of the shuttle vectors described above and to introduce such ahybrid vectors into strains of Corynebacterium glutamicum.Transformation of C. glutamicum can be achieved by protoplasttransformation (Kastsumata, R. et al. (1984) J. Bacteriol. 159306-311),electroporation (Liebl, E. et al. (1989) FEMS Microbiol. Letters,53:399-303) and in cases where special vectors are used, also byconjugation (as described e.g. in Schafer, A et al. (1990) J. Bacteriol.172:1663-1666). It is also possible to transfer the shuttle vectors forC. glutamicum to E. coli by preparing plasmid DNA from C. glutamicum(using standard methods well-known in the art) and transforming it intoE. coli. This transformation step can be performed using standardmethods, but it is advantageous to use an Mcr-deficient E. coli strain,such as NM522 (Gough & Murray (1983) J. Mol. Biol. 166:1-19).

Genes may be overexpressed in C. glutamicum strains using plasmids whichcomprise pCG1 (U.S. Pat. No. 4,617,267) or fragments thereof, andoptionally the gene for kanamycin resistance from TN903 (Grindley, N. D.and Joyce, C. M. (1980) Proc. Natl. Acad. Sci. USA 77(12): 7176-7180).In addition, genes may be overexpressed in C. glutamicum strains usingplasmid pSL109 (Lee, H.-S. and A. J. Sinskey (1994) J. Microbiol.Biotechnol. 4: 256-263).

Aside from the use of replicative plasmids, gene overexpression can alsobe achieved by integration into the genome. Genomic integration in C.glutamicum or other Corynebacterium or Brevibacterium species may beaccomplished by well-known methods, such as homologous recombinationwith genomic region(s), restriction endonuclease mediated integration(REMI) (see, e.g., DE Patent 19823834), or through the use oftransposons. It is also possible to modulate the activity of a gene ofinterest by modifying the regulatory regions (e.g., a promoter, arepressor, and/or an enhancer) by sequence modification, insertion, ordeletion using site-directed methods (such as homologous recombination)or methods based on random events (such as transposon mutagenesis orREMI). Nucleic acid sequences which function as transcriptionalterminators may also be inserted 3′ to the coding region of one or moregenes of the invention; such terminators are well-known in the art andare described, for example, in Winnacker, E. L. (1987) From Genes toClones—Introduction to Gene Technology. VCH: Weinheim.

Example 6 Assessment of the Expression of the Mutant Protein

Observations of the activity of a mutated protein in a transformed hostcell rely on the fact that the mutant protein is expressed in a similarfashion and in a similar quantity to that of the wild-type protein. Auseful method to ascertain the level of transcription of the mutant gene(an indicator of the amount of mRNA available for translation to thegene product) is to perform a Northern blot (for reference see, forexample, Ausubel et al. (1988) Current Protocols in Molecular Biology,Wiley: New York), in which a primer designed to bind to the gene ofinterest is labeled with a detectable tag (usually radioactive orchemiluminescent), such that when the total RNA of a culture of theorganism is extracted, run on gel, transferred to a stable matrix andincubated with this probe, the binding and quantity of binding of theprobe indicates the presence and also the quantity of mRNA for thisgene. This information is evidence of the degree of transcription of themutant gene. Total cellular RNA can be prepared from Corynebacteriumglutamicum by several methods, all well-known in the art, such as thatdescribed in Bormann, E. R. et al. (1992) Mol. Microbiol. 6: 317-326.

To assess the presence or relative quantity of protein translated fromthis mRNA, standard techniques, such as a Western blot, may be employed(see, for example, Ausubel et al. (1988) Current Protocols in MolecularBiology, Wiley: N.Y.). In this process, total cellular proteins areextracted, separated by gel electrophoresis, transferred to a matrixsuch as nitrocellulose, and incubated with a probe, such as an antibody,which specifically binds to the desired protein. This probe is generallytagged with a chemiluminescent or colorimetric label which may bereadily detected. The presence and quantity of label observed indicatesthe presence and quantity of the desired mutant protein present in thecell.

Example 7 Growth of Genetically Modified Corynebacteriumglutamicum—Media and Culture Conditions

Genetically modified Corynebacteria are cultured in synthetic or naturalgrowth media. A number of different growth media for Corynebacteria areboth well-known and readily available (Lieb et al. (1989) Appl.Microbiol. Biotechnol., 32:205-210; von der Osten et al. (1998)Biotechnology Letters, 11:11-16; Patent DE 4,120,867; Liebl (1992) “TheGenus Corynebacterium , in: The Procaryotes, Volume II, Balows, A. etal., eds. Springer-Verlag). These media consist of one or more carbonsources, nitrogen sources, inorganic salts, vitamins and trace elements.Preferred carbon sources are sugars, such as mono-, di-, orpolysaccharides. For example, glucose, fructose, mannose, galactose,ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starchor cellulose serve as very good carbon sources. It is also possible tosupply sugar to the media via complex compounds such as molasses orother by-products from sugar refinement. It can also be advantageous tosupply mixtures of different carbon sources. Other possible carbonsources are alcohols and organic acids, such as methanol, ethanol,acetic acid or lactic acid. Nitrogen sources are usually organic orinorganic nitrogen compounds, or materials which contain thesecompounds. Exemplary nitrogen sources include ammonia gas or ammoniasalts, such as NH₄Cl or (NH₄)₂SO₄, NH₄OH, nitrates, urea, amino acids orcomplex nitrogen sources like corn steep liquor, soy bean flour, soybean protein, yeast extract, meat extract and others.

Inorganic salt compounds which may be included in the media include thechloride-, phosphorous- or sulfate-salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.Chelating compounds can be added to the medium to keep the metal ions insolution. Particularly useful chelating compounds includedihydroxyphenols, like catechol or protocatechuate, or organic acids,such as citric acid. It is typical for the media to also contain othergrowth factors, such as vitamins or growth promoters, examples of whichinclude biotin, riboflavin, thiamin, folic acid, nicotinic acid,pantothenate and pyridoxin. Growth factors and salts frequentlyoriginate from complex media components such as yeast extract, molasses,corn steep liquor and others. The exact composition of the mediacompounds depends strongly on the immediate experiment and isindividually decided for each specific case. Information about mediaoptimization is available in the textbook “Applied Microbiol.Physiology, A Practical Approach (eds. P. M. Rhodes, P. F. Stanbury, IRLPress (1997) pp. 53-73, ISBN 0 19 963577 3). It is also possible toselect growth media from commercial suppliers, like standard 1 (Merck)or BHI (grain heart infusion, DIFCO) or others.

All medium components are sterilized, either by heat (20 minutes at 1.5bar and 121° C.) or by sterile filtration. The components can either besterilized together or, if necessary, separately. All media componentscan be present at the beginning of growth, or they can optionally beadded continuously or batchwise.

Culture conditions are defined separately for each experiment. Thetemperature should be in a range between 15° C. and 45° C. Thetemperature can be kept constant or can be altered during theexperiment. The pH of the medium should be in the range of 5 to 8.5,preferably around 7.0, and can be maintained by the addition of buffersto the media. An exemplary buffer for this purpose is a potassiumphosphate buffer. Synthetic buffers such as MOPS, HEPES, ACES and otherscan alternatively or simultaneously be used. It is also possible tomaintain a constant culture pH through the addition of NaOH or NH₄OHduring growth. If complex medium components such as yeast extract are:utilized, the necessity for additional buffers may be reduced, due tothe fact that many complex compounds have high buffer capacities. If afermentor is utilized for culturing the microorganisms, the pH can alsobe controlled using gaseous ammonia.

The incubation time is usually in a range from several hours to severaldays. This time is selected in order to permit the maximal amount ofproduct to accumulate in the broth. The disclosed growth experiments canbe carried out in a variety of vessels, such as microtiter plates, glasstubes, glass flasks or glass or metal fermentors of different sizes. Forscreening a large number of clones, the microorganisms should becultured in microtiter plates, glass tubes or shake flasks, either withor without baffles. Preferably 100 ml shake flasks are used, filled with10% (by volume) of the required growth medium. The flasks should beshaken on a rotary shaker (amplitude 25 mm) using a speed-range of100-300 rpm. Evaporation losses can be diminished by the maintenance ofa humid atmosphere; alternatively, a mathematical correction forevaporation losses should be performed.

If genetically modified clones are tested, an unmodified control cloneor a control clone containing the basic plasmid without any insertshould also be tested. The medium is inoculated to an OD₆₀₀ of 0.5-1.5using cells grown on agar plates, such as CM plates (10 g/l glucose, 2.5g/l NaCl, 2 g/l urea, 10 g/l polypeptone, 5 g/l yeast extract, 5 g/lmeat extract, 22 g/l NaCl, 2 g/l urea, 10 g/l polypeptone, 5 g/l yeastextract, 5 g/l meat extract, 22 g/l agar, pH 6.8 with 2M NaOH) that hadbeen incubated at 30° C. Inoculation of the media is accomplished byeither introduction of a saline suspension of C. glutamicum cells fromCM plates or addition of a liquid preculture of this bacterium.

Example 8 In Vitro Analysis of the Function of Mutant Proteins

The determination of activities and kinetic parameters of enzymes iswell established in the art. Experiments to determine the activity ofany given altered enzyme must be tailored to the specific activity ofthe wild-type enzyme, which is well within the ability of one ofordinary skill in the art. Overviews about enzymes in general, as wellas specific details concerning structure, kinetics, principles, methods,applications and examples for the determination of many enzymeactivities may be found, for example, in the following references:Dixon, M., and Webb, E. C., (1979) Enzymes. Longmans: London; Fersht,(1985) Enzyme Structure and Mechanism. Freeman: New York; Walsh, (1979)Enzymatic Reaction Mechanisms. Freeman: San Francisco; Price, N. C.,Stevens, L. (1982) Fundamentals of Enzymology. Oxford Univ. Press:Oxford; Boyer, P. D., ed. (1983) The Enzymes, 3^(rd) ed. Academic Press:New York; Bisswanger, H., (1994) Enzymkinetik, 2^(nd) ed. VCH: Weinheim(ISBN 3527300325); Bergmeyer, H. U., Bergmeyer, J., Graβl, M., eds.(1983-1986) Methods of Enzymatic Analysis, 3^(rd) ed., vol. I-XII,Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of IndustrialChemistry (1987) vol. A9, “Enzymes”. VCH: Weinheim, p. 352-363.

The activity of proteins which bind to DNA can be measured by severalwell-established methods, such as DNA band-shift assays (also called gelretardation assays). The effect of such proteins on the expression ofother molecules can be measured using reporter gene assays (such as thatdescribed in Kolmar, H. et al. (1995) EMBO J. 14: 3895-3904 andreferences cited therein). Reporter gene test systems are well known andestablished for applications in both pro- and eukaryotic cells, usingenzymes such as beta-galactosidase, green fluorescent protein, andseveral others.

The determination of activity of membrane-transport proteins can beperformed according to techniques such as those described in Gennis, R.B. (1989) “Pores, Channels and Transporters”, in Biomembranes, MolecularStructure and Function, Springer: Heidelberg, p. 85-137; 199-234; and270-322.

Example 9 Analysis of Impact of Mutant Protein on the Production of theDesired Product

The effect of the genetic modification in C. glutamicum on production ofa desired compound (such as an amino acid) can be assessed by growingthe modified microorganism under suitable conditions (such as thosedescribed above) and analyzing the medium and/or the cellular componentfor increased production of the desired product (i.e., an amino acid).Such analysis techniques are well known to one of ordinary skill in theart, and include spectroscopy, thin layer chromatography, stainingmethods of various kinds, enzymatic and microbiological methods, andanalytical chromatography such as high performance liquid chromatography(see, for example, Ullman, Encyclopedia of Industrial Chemistry, vol.A2, p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A. et al.,(1987) “Applications of HPLC in Biochemistry” in: Laboratory Techniquesin Biochemistry and Molecular Biology, vol. 17; Rehm et al. (1993)Biotechnology, vol. 3, Chapter III: “Product recovery and purification”,page 469-714, VCH: Weinheim; Belter, P. A. et al. (1988) Bioseparations:downstream processing for biotechnology, John Wiley and Sons; Kennedy,J. F. and Cabral, J. M. S. (1992) Recovery processes for biologicalmaterials, John Wiley and Sons; Shaeiwitz, J. A. and Henry, J. D. (1988)Biochemical separations, in: Ulmann's Encyclopedia of IndustrialChemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, NoyesPublications.)

In addition to the measurement of the final product of fermentation, itis also possible to analyze other components of the metabolic pathwaysutilized for the production of the desired compound, such asintermediates and side-products, to determine the overall efficiency ofproduction of the compound. Analysis methods include measurements ofnutrient levels in the medium (e.g., sugars, hydrocarbons, nitrogensources, phosphate, and other ions), measurements of biomass compositionand growth, analysis of the production of common metabolites ofbiosynthetic pathways, and measurement of gasses produced duringfermentation. Standard methods for these measurements are outlined inApplied Microbial Physiology, A Practical Approach, P. M. Rhodes and P.F. Stanbury, eds., IRL Press, p. 103-129; 131-163; and 165-192 (ISBN:0199635773) and references cited therein.

Example 10 Purification of the Desired Product from C. glutamicumCulture

Recovery of the desired product from the C. glutamicum cells orsupernatant of the above-described culture can be performed by variousmethods well known in the art. If the desired product is not secretedfrom the cells, the cells can be harvested from the culture by low-speedcentrifugation, the cells can be lysed by standard techniques, such asmechanical force or sonication. The cellular debris is removed bycentrifugation, and the supernatant fraction containing the solubleproteins is retained for further purification of the desired compound.If the product is secreted from the C. glutamicum cells, then the cellsare removed from the culture by low-speed centrifugation, and thesupernate fraction is retained for further purification.

The supernatant fraction from either purification method is subjected tochromatography with a suitable resin, in which the desired molecule iseither retained on a chromatography resin while many of the impuritiesin the sample are not, or where the impurities are retained by the resinwhile the sample is not. Such chromatography steps may be repeated asnecessary, using the same or different chromatography resins. One ofordinary skill in the art would be well-versed in the selection ofappropriate chromatography resins and in their most efficaciousapplication for a particular molecule to be purified. The purifiedproduct may be concentrated by filtration or ultrafiltration, and storedat a temperature at which the stability of the product is maximized.

There are a wide array of purification methods known to the art and thepreceding method of purification is not meant to be limiting. Suchpurification techniques are described, for example, in Bailey, J. E. &Ollis, D. F. Biochemical Engineering Fundamentals, McGraw-Hill: New York(1986).

The identity and purity of the isolated compounds may be assessed bytechniques standard in the art. These include high-performance liquidchromatography (HPLC), spectroscopic methods, staining methods, thinlayer chromatography, NIRS, enzymatic assay, or microbiologically. Suchanalysis methods are reviewed in: Patek et al. (1994) Appl. Environ.Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11:27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70.Ulmann's Encyclopedia of Industrial Chemistry, (1996) vol. A27, VCH:Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566, 575-581 and p.581-587; Michal, G. (1999) Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. etal. (1987) Applications of HPLC in Biochemistry in: LaboratoryTechniques in Biochemistry and Molecular Biology, vol. 17.

Example 11 Analysis of the Gene Sequences of the Invention

The comparison of sequences and determination of percent homologybetween two sequences are art-known techniques, and can be accomplishedusing a mathematical algorithm, such as the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as inKarlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Suchan algorithm is incorporated into the NBLAST and XBLAST programs(version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to SMP nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to SMP protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, one ofordinary skill in the art will know how to optimize the parameters ofthe program (e.g., XBLAST and NBLAST) for the specific sequence beinganalyzed.

Another example of a mathematical algorithm utilized for the comparisonof sequences is the algorithm of Meyers and Miller ((1988) Comput. Appl.Biosci. 4: 11-17). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) which is part of the GCG sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 can be used. Additional algorithms forsequence analysis are known in the art, and include ADVANCE and ADAM,described in Torelli and Robotti (1994) Comput. Appl. Biosci. 10:3-5;and FASTA, described in Pearson and Lipman (1988) P.N.A.S. 85:2444-8.

The percent homology between two amino acid sequences can also beaccomplished using the GAP program in the GCG software package(available at http://www.gcg.com), using either a Blosum 62 matrix or aPAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a lengthweight of 2, 3, or 4. The percent homology between two nucleic acidsequences can be accomplished using the GAP program in the GCG softwarepackage, using standard parameters, such as a gap weight of 50 and alength weight of 3.

A comparative analysis of the gene sequences of the invention with thosepresent in Genbank has been performed using techniques known in the art(see, e.g., Bexevanis and Ouellette, eds. (1998) Bioinformatics: APractical Guide to the Analysis of Genes and Proteins. John Wiley andSons: New York). The gene sequences of the invention were compared togenes present in Genbank in a three-step process. In a first step, aBLASTN analysis (e.g., a local alignment analysis) was performed foreach of the sequences of the invention against the nucleotide sequencespresent in Genbank, and the top 500 hits were retained for furtheranalysis. A subsequent FASTA search (e.g., a combined local and globalalignment analysis, in which limited regions of the sequences arealigned) was performed on these 500 hits. Each gene sequence of theinvention was subsequently globally aligned to each of the top threeFASTA hits, using the GAP program in the GCG software package (usingstandard parameters). In order to obtain correct results, the length ofthe sequences extracted from Genbank were adjusted to the length of thequery sequences by methods well-known in the art. The results of thisanalysis are set forth in Table 4. The resulting data is identical tothat which would have been obtained had a GAP (global) analysis alonebeen performed on each of the genes of the invention in comparison witheach of the references in Genbank, but required significantly reducedcomputational time as compared to such a database-wide GAP (global)analysis. Sequences of the invention for which no alignments above thecutoff values were obtained are indicated on Table 4 by the absence ofalignment information. It will further be understood by one of ordinaryskill in the art that the GAP alignment homology percentages set forthin Table 4 under the heading “% homology (GAP)” are listed in theEuropean numerical format, wherein a ‘,’ represents a decimal point. Forexample, a value of “40,345” in this column represents “40.345%”.

Example 12 Construction and Operation of DNA Microarrays

The sequences of the invention may additionally be used in theconstruction and application of DNA microarrays (the design,methodology, and uses of DNA arrays are well known in the art, and aredescribed, for example, in Schena, M. et al. (1995) Science 270:467-470; Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359-1367;DeSaizieu, A. et al. (1998) Nature Biotechnology 16: 45-48; and DeRisi,J. L. et al. (1997) Science 278: 680-686).

DNA microarrays are solid or flexible supports consisting ofnitrocellulose, nylon, glass, silicone, or other materials. Nucleic acidmolecules may be attached to the surface in an ordered manner. Afterappropriate labeling, other nucleic acids or nucleic acid mixtures canbe hybridized to the immobilized nucleic acid molecules, and the labelmay be used to monitor and measure the individual signal intensities ofthe hybridized molecules at defined regions. This methodology allows thesimultaneous quantification of the relative or absolute amount of all orselected nucleic acids in the applied nucleic acid sample or mixture.DNA microarrays, therefore, permit an analysis of the expression ofmultiple (as many as 6800 or more) nucleic acids in parallel (see, e.g.,Schena, M. (1996) BioEssays 18(5): 427-431).

The sequences of the invention may be used to design oligonucleotideprimers which are able to amplify defined regions of one or more C.glutamicum genes by a nucleic acid amplification reaction such as thepolymerase chain reaction. The choice and design of the 5′ or 3′oligonucleotide primers or of appropriate linkers allows the covalentattachment of the resulting PCR products to the surface of a supportmedium described above (and also described, for example, Schena, M. etal. (1995) Science 270: 467-470).

Nucleic acid microarrays may also be constructed by in situoligonucleotide synthesis as described by Wodicka, L. et al. (1997)Nature Biotechnology 15: 1359-1367. By photolithographic methods,precisely defined regions of the matrix are exposed to light. Protectivegroups which are photolabile are thereby activated and undergonucleotide addition, whereas regions that are masked from light do notundergo any modification. Subsequent cycles of protection and lightactivation permit the synthesis of different oligonucleotides at definedpositions. Small, defined regions of the genes of the invention may besynthesized on microarrays by solid phase oligonucleotide synthesis.

The nucleic acid molecules of the invention present in a sample ormixture of nucleotides may be hybridized to the microarrays. Thesenucleic acid molecules can be labeled according to standard methods. Inbrief, nucleic acid molecules (e.g., mRNA molecules or DNA molecules)are labeled by the incorporation of isotopically or fluorescentlylabeled nucleotides, e.g., during reverse transcription or DNAsynthesis. Hybridization of labeled nucleic acids to microarrays isdescribed (e.g., in Schena, M. et al. (1995) supra; Wodicka, L. et al.(1997), supra; and DeSaizieu A. et al. (1998), supra). The detection andquantification of the hybridized molecule are tailored to the specificincorporated label. Radioactive labels can be detected, for example, asdescribed in Schena, M. et al. (1995) supra) and fluorescent labels maybe detected, for example, by the method of Shalon et al. (1996) GenomeResearch 6: 639-645).

The application of the sequences of the invention to DNA microarraytechnology, as described above, permits comparative analyses ofdifferent strains of C. glutamicum or other Corynebacteria. For example,studies of inter-strain variations based on individual transcriptprofiles and the identification of genes that are important for specificand/or desired strain properties such as pathogenicity, productivity andstress tolerance are facilitated by nucleic acid array methodologies.Also, comparisons of the profile of expression of genes of the inventionduring the course of a fermentation reaction are possible using nucleicacid array technology.

Example 13 Analysis of the Dynamics of Cellular Protein Populations(Proteomics)

The genes, compositions, and methods of the invention may be applied tostudy the interactions and dynamics of populations of proteins, termed‘proteomics’. Protein populations of interest include, but are notlimited to, the total protein population of C. glutamicum (e.g., incomparison with the protein populations of other organisms), thoseproteins which are active under specific environmental or metabolicconditions (e.g., during fermentation, at high or low temperature, or athigh or low pH), or those proteins which are active during specificphases of growth and development.

Protein populations can be analyzed by various well-known techniques,such as gel electrophoresis. Cellular proteins may be obtained, forexample, by lysis or extraction, and may be separated from one anotherusing a variety of electrophoretic techniques. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) separates proteins largelyon the basis of their molecular weight. Isoelectric focusingpolyacrylamide gel electrophoresis (IEF-PAGE) separates proteins bytheir isoelectric point (which reflects not only the amino acid sequencebut also posttranslational modifications of the protein). Another, morepreferred method of protein analysis is the consecutive combination ofboth IEF-PAGE and SDS-PAGE, known as 2-D-gel electrophoresis (described,for example, in Hermann et al. (1998) Electrophoresis 19: 3217-3221;Fountoulakis et al. (1998) Electrophoresis 19: 1193-1202; Langen et al.(1997) Electrophoresis 18: 1184-1192; Antelmann et al. (1997)Electrophoresis 18: 1451-1463). Other separation techniques may also beutilized for protein separation, such as capillary gel electrophoresis;such techniques are well known in the art.

Proteins separated by these methodologies can be visualized by standardtechniques, such as by staining or labeling. Suitable stains are knownin the art, and include Coomassie Brilliant Blue, silver stain, orfluorescent dyes such as Sypro Ruby (Molecular Probes). The inclusion ofradioactively labeled amino acids or other protein precursors (e.g.,³⁵S-methionine, ³⁵S-cysteine, ¹⁴C-labelled amino acids, ¹⁵N-amino acids,¹⁵NO₃ or ¹⁵NH₄ ^(+ or) ¹³C-labelled amino acids) in the medium of C.glutamicum permits the labeling of proteins from these cells prior totheir separation. Similarly, fluorescent labels may be employed. Theselabeled proteins can be extracted, isolated and separated according tothe previously described techniques.

Proteins visualized by these techniques can be further analyzed bymeasuring the amount of dye or label used. The amount of a given proteincan be determined quantitatively using, for example, optical methods andcan be compared to the amount of other proteins in the same gel or inother gels. Comparisons of proteins on gels can be made, for example, byoptical comparison, by spectroscopy, by image scanning and analysis ofgels, or through the use of photographic films and screens. Suchtechniques are well-known in the art.

To determine the identity of any given protein, direct sequencing orother standard techniques may be employed. For example, N- and/orC-terminal amino acid sequencing (such as Edman degradation) may beused, as may mass spectrometry (in particular MALDI or ESI techniques(see, e.g., Langen et al. (1997) Electrophoresis 18: 1184-1192)). Theprotein sequences provided herein can be used for the identification ofC. glutamicum proteins by these techniques.

The information obtained by these methods can be used to comparepatterns of protein presence, activity, or modification betweendifferent samples from various biological conditions (e.g., differentorganisms, time points of fermentation, media conditions, or differentbiotopes, among others). Data obtained from such experiments alone, orin combination with other techniques, can be used for variousapplications, such as to compare the behavior of various organisms in agiven (e.g., metabolic) situation, to increase the productivity ofstrains which produce fine chemicals or to increase the efficiency ofthe production of fine chemicals.

Equivalents

Those of ordinary skill in the art will recognize, or will be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

1. An isolated nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:179, or the complement thereof.
 2. An isolated nucleic acidmolecule comprising a nucleotide sequence which is at least 95%identical to the entire nucleotide sequence of SEQ ID NO:179, whereinthe nucleic acid molecule encodes a polypeptide having aphosphoenolpyruvate carboxykinase activity, or the complement thereof.3. An isolated nucleic acid molecule comprising a nucleotide sequencewhich is at least 98% identical to the entire nucleotide sequence of SEQID NO:179, wherein the nucleic acid molecule encodes a polypeptidehaving a phosphoenolpyruvate carboxykinase activity, or the complementthereof.
 4. An isolated nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:180, or thecomplement thereof.
 5. An isolated nucleic acid molecule comprising afragment of at least 25 contiguous nucleotides of the nucleotidesequence of SEQ ID NO:179, wherein the fragment encodes a polypeptidehaving a phosphoenolpyruvate carboxykinase activity, or the complementthereof.
 6. An isolated nucleic acid molecule consisting of a fragmentof at least 25 contiguous nucleotides of the nucleotide sequence of SEQID NO:179, or the complement thereof.
 7. An isolated nucleic acidmolecule which encodes a polypeptide comprising an amino acid sequencewhich is at least 95% identical to the entire amino acid sequence of SEQID NO:180, wherein the polypeptide has a phosphoenolpyruvatecarboxykinase activity, or the complement thereof.
 8. An isolatednucleic acid molecule which encodes a polypeptide comprising an aminoacid sequence which is at least 98% identical to the entire amino acidsequence of SEQ ID NO:180, wherein the polypeptide has aphosphoenolpyruvate carboxykinase activity, or the complement thereof.9. An isolated nucleic acid molecule comprising the nucleic acidmolecule of any one of claims 1 or 2-8 and a nucleotide sequenceencoding a heterologous polypeptide.
 10. A vector comprising the nucleicacid molecule of any one of claims 1 or 2-8.
 11. The vector of claim 10,which is an expression vector.
 12. A host cell transfected with theexpression vector of claim 11, wherein said host cell is amicroorganism.
 13. The host cell of claim 12, wherein said cell is abacterial cell.
 14. The host cell of claim 13, wherein said cell belongsto the genus Corynebacterium or Brevibactertum.
 15. A method ofproducing a polypeptide comprising culturing the host cell of claim 12in an appropriate culture medium to, thereby, produce the polypeptide.16. A method for producing an amino acid, comprising culturing the cellof claim 12 such that the amino acid is produced.
 17. The method ofclaim 16, wherein said method further comprises the step of recoveringthe amino acid from said culture.
 18. The method of claim 16, whereinsaid cell belongs to the genus Corynebactertium or Brevibacterium. 19.The method of claim 16, wherein said cell is selected from the groupconsisting of Corynebacterium glutamicum, Corynebacterium herculis,Corynebacterium lilium, Corynebactertium acetoacidophilum,Corynebacterium acetoglutamicum, Corynebacterium acetophilum,Corynebacterium ammoniagenes, Corynebacterium fujiokense,Corynebacterium nitrilophilus, Brevibacterium ammoniagenes,Brevibacterium butanicum, Brevibacterium divaricatum, Brevibacteriumflavum, Brevibacterium healii, Brevibacterium ketoglutamicum,Brevibacterium ketosoreductum, Brevibacterium lactofermentum,Brevibacterium linens, Brevibacterium paraffinolyticum, and thosestrains set forth in Table
 3. 20. The metbod of claim 16, whereinexpression of the nucleic acid molecule from said vector results inmodulation of production of said amino acid.
 21. The method of claim 16,wherein said anino acid is selected from the group consisting of lysine,glutamate, glutamine, alanine, aspartate, glycine, serine, threonine,methionine, cysteine, valine, leucine, isoleucine, arginine, proline,histidine, tyrosine, phenylalanine, and tryptophan.
 22. A method fordiagnosing the presence or activity of Corynebacterium diphtheriae in asubject, comprising detecting the presence of at least one of thenucleic acid molecules of any one of claims 1 or 2-8, thereby diagnosingthe presence or activity of Corynebacterium diphtheriae in the subject.